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1
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Agarwal S, Taft E, Gauthier M, Darcy J, Buckowing K, Berman D, Davis WP, Rogers AB, Janas MM. Mechanistic Insights into Hybridization-Based Off-Target Activity of GalNAc-siRNA Conjugates. Nucleic Acid Ther 2025. [PMID: 40134378 DOI: 10.1089/nat.2024.0090] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/27/2025] Open
Abstract
Nonclinical safety screening of small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand is typically carried out in rats at exaggerated exposures in a repeat-dose regimen. We have previously shown that at these suprapharmacological doses, hepatotoxicity observed with a subset of GalNAc-siRNAs is largely driven by undesired RNA-induced silencing complex (RISC)-mediated antisense strand seed-based off-target activity, similar to microRNA-like regulation. However, the RISC component requirements for off-target activity of siRNAs have not been evaluated. Here, we evaluate the roles of major RISC components, AGO and TNRC6 (or GW182) proteins, in driving on- and off-target activity of GalNAc-siRNAs in hepatocytes, in vitro and in vivo. We demonstrate that knocking down AGO2, but not AGO1 or AGO4, is protective against GalNAc-siRNA-driven off-target activity and hepatotoxicity. As expected, knocking down AGO2, but not AGO1 or AGO4, reduces the on-target activity of GalNAc-siRNA. Similarly, knocking down TNRC6 paralogs, TNRC6A or TNRC6B, but not TNRC6C, is protective against off-target activity and hepatotoxicity while having minimal impact on the on-target activity of GalNAc-siRNA. These data indicate that while AGO2 is the only RISC component required for the on-target activity of GalNAc-siRNAs, the undesired off-target activity and hepatotoxicity of a subset of GalNAc-siRNAs are mediated via the RISC composed predominantly of AGO2 and TNRC6 paralogs TNRC6A and/or TNRC6B.
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Affiliation(s)
- Saket Agarwal
- Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA
| | | | | | - Justin Darcy
- Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA
| | | | - Daniel Berman
- Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA
| | | | | | - Maja M Janas
- Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA
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2
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Moescheid MF, Puckelwaldt O, Beutler M, Haeberlein S, Grevelding CG. Defining an optimal control for RNAi experiments with adult Schistosoma mansoni. Sci Rep 2023; 13:9766. [PMID: 37328492 PMCID: PMC10276032 DOI: 10.1038/s41598-023-36826-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Accepted: 06/10/2023] [Indexed: 06/18/2023] Open
Abstract
In parasites such as Schistosoma mansoni, gene knockdown by RNA interference (RNAi) has become an indispensable tool for functional gene characterization. To distinguish target-specific RNAi effects versus off-target effects, controls are essential. To date, however, there is still no general agreement about suitable RNAi controls, which limits the comparability between studies. To address this point, we investigated three selected dsRNAs for their suitability as RNAi controls in experiments with adult S. mansoni in vitro. Two dsRNAs were of bacterial origin, the neomycin resistance gene (neoR) and the ampicillin resistance gene (ampR). The third one, the green fluorescent protein gene (gfp), originated from jellyfish. Following dsRNA application, we analyzed physiological parameters like pairing stability, motility, and egg production as well as morphological integrity. Furthermore, using RT-qPCR we evaluated the potential of the used dsRNAs to influence transcript patterns of off-target genes, which had been predicted by si-Fi (siRNA-Finder). At the physiological and morphological levels, we observed no obvious changes in the dsRNA treatment groups compared to an untreated control. However, we detected remarkable differences at the transcript level of gene expression. Amongst the three tested candidates, we suggest dsRNA of the E. coli ampR gene as the most suitable RNAi control.
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Affiliation(s)
- Max F Moescheid
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Giessen, Germany
| | - Oliver Puckelwaldt
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Giessen, Germany
| | - Mandy Beutler
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Giessen, Germany
| | - Simone Haeberlein
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Giessen, Germany
| | - Christoph G Grevelding
- Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, Giessen, Germany.
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Bharathi JK, Anandan R, Benjamin LK, Muneer S, Prakash MAS. Recent trends and advances of RNA interference (RNAi) to improve agricultural crops and enhance their resilience to biotic and abiotic stresses. PLANT PHYSIOLOGY AND BIOCHEMISTRY : PPB 2023; 194:600-618. [PMID: 36529010 DOI: 10.1016/j.plaphy.2022.11.035] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/16/2022] [Revised: 11/04/2022] [Accepted: 11/26/2022] [Indexed: 06/17/2023]
Abstract
Over the last two decades, significant advances have been made using genetic engineering technology to modify genes from various exotic origins and introduce them into plants to induce favorable traits. RNA interference (RNAi) was discovered earlier as a natural process for controlling the expression of genes across all higher species. It aims to enhance precision and accuracy in pest/pathogen resistance, quality improvement, and manipulating the architecture of plants. However, it existed as a widely used technique recently. RNAi technologies could well be used to down-regulate any genes' expression without disrupting the expression of other genes. The use of RNA interference to silence genes in various organisms has become the preferred method for studying gene functions. The establishment of new approaches and applications for enhancing desirable characters is essential in crops by gene suppression and the refinement of knowledge of endogenous RNAi mechanisms in plants. RNAi technology in recent years has become an important and choicest method for controlling insects, pests, pathogens, and abiotic stresses like drought, salinity, and temperature. Although there are certain drawbacks in efficiency of this technology such as gene candidate selection, stability of trigger molecule, choice of target species and crops. Nevertheless, from past decade several target genes has been identified in numerous crops for their improvement towards biotic and abiotic stresses. The current review is aimed to emphasize the research done on crops under biotic and abiotic stress using RNAi technology. The review also highlights the gene regulatory pathways/gene silencing, RNA interference, RNAi knockdown, RNAi induced biotic and abiotic resistance and advancements in the understanding of RNAi technology and the functionality of various components of the RNAi machinery in crops for their improvement.
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Affiliation(s)
- Jothi Kanmani Bharathi
- Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalai Nagar, 608 002, Tamil Nadu, India
| | - Ramaswamy Anandan
- Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalai Nagar, 608 002, Tamil Nadu, India
| | - Lincy Kirubhadharsini Benjamin
- Horticulture and Molecular Physiology Lab, School of Agricultural Innovations and Advanced Learning, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India
| | - Sowbiya Muneer
- Horticulture and Molecular Physiology Lab, School of Agricultural Innovations and Advanced Learning, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India.
| | - Muthu Arjuna Samy Prakash
- Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalai Nagar, 608 002, Tamil Nadu, India.
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Chai H, Lei Z, Liu Y, Gong J, Cao Z, Huang Z, Yang H, Wu Z. miR-505-5p alleviates acute rejection of liver transplantation by inhibiting Myd88 and inducing M2 polarizationof Kupffer cells. Acta Biochim Biophys Sin (Shanghai) 2022; 54:1148-1158. [PMID: 35959879 PMCID: PMC9828294 DOI: 10.3724/abbs.2022100] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2022] [Accepted: 06/08/2022] [Indexed: 11/25/2022] Open
Abstract
The occurrence of acute rejection after liver transplantation seriously impairs the prognosis of patients. miRNA is involved in many physiological and pathological processes of the body, but the mechanism of miRNA action in liver transplantation is not completely clear. In this study, we discuss the role of miR-505-5p in acute rejection after liver transplantation and its putative regulating mechanism. We construct an allogeneic rat liver transplantation model, observe the morphological and pathological changes in liver tissue, detect the expression levels of Myd88, miR-505-5p, IL-10 and TNF-α, and confirm that Myd88 is one of the direct targets of miR-505. The effects of miR-505-5p on the Myd88/TRAF6/NF-κB and MAPK pathways are detected both in vitro and in vivo, and the standard markers of Kupffer cell M1/M2 polarization are also detected. The results of qRT-PCR experiments show that miR-505-5p has a downward trend in rats with acute rejection. Western blot analysis reveals that over-expression of miR-505-5p induces the reduction of NF-κB and MAPK pathways both in vitro and in vivo. The role of miR-505-5p in alleviating acute rejection after transplantation may be accomplished by inducing M2-type polarization of Kupffer cells. In conclusion, we find that miR-505-5p alleviates acute rejection of liver transplantation by inducing M2 polarization of macrophages via the Myd88/TRAF6 axis, which suggests a potential strategy based on miRNAs in the follow-up treatment of liver transplantation.
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Affiliation(s)
- Hao Chai
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Zilun Lei
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Yanyao Liu
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Junhua Gong
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Zhenrui Cao
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Zuotian Huang
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Hang Yang
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
| | - Zhongjun Wu
- Department of Hepatobiliary Surgerythe First Affiliated Hospital of Chongqing Medical UniversityChongqing400042China
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Hauptmann J, Hehne V, Balzer M, Bethge L, Wikstrom Lindholm M. Engineering miRNA features into siRNAs: Guide-strand bulges are compatible with gene repression. MOLECULAR THERAPY - NUCLEIC ACIDS 2022; 27:1116-1126. [PMID: 35251767 PMCID: PMC8881630 DOI: 10.1016/j.omtn.2022.02.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Accepted: 02/06/2022] [Indexed: 11/22/2022]
Abstract
Synthetic siRNA guide strands are typically designed with perfect complementarity to the passenger strand and the target mRNA. We examined whether siRNAs with intentional guide-strand bulges are functional in vitro and in vivo. Importantly, this was done by systematic shortening of the passenger strand, evaluating identical 19-mer guide-strand sequences but forcing them into conformations with 1- to 4-nt bulges after annealing. We demonstrate that guide-strand bulges can be well tolerated at several positions of unmodified and modified siRNAs. Beyond that, we show that GalNAc-conjugated siRNAs with bulges at certain positions of the guide strand repress transthyretin in murine primary hepatocytes and in vivo in mice. In vivo, a GalNAc-conjugated siRNA with a 1-nt bulge at position 14 of the guide strand was as active as the perfectly complementary siRNA. Finally, in a luciferase reporter system, mRNA target sequences were systematically shortened so that RNA-induced silencing complex activity could only occur with a guide-strand bulge. Here, luciferase reporters were repressed when 1- and 2-nt deletions of the reporter were applied to the edges of the sequence. We conclude that some guide-strand bulges versus target transcript can result in target repression and therefore should be evaluated as off-target risks.
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6
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Kilikevicius A, Meister G, Corey DR. Reexamining assumptions about miRNA-guided gene silencing. Nucleic Acids Res 2022; 50:617-634. [PMID: 34967419 PMCID: PMC8789053 DOI: 10.1093/nar/gkab1256] [Citation(s) in RCA: 117] [Impact Index Per Article: 39.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 12/01/2021] [Accepted: 12/06/2021] [Indexed: 12/16/2022] Open
Abstract
MicroRNAs (miRNAs) are short endogenously expressed RNAs that have the potential to regulate the expression of any RNA. This potential has led to the publication of several thousand papers each year connecting miRNAs to many different genes and human diseases. By contrast, relatively few papers appear that investigate the molecular mechanism used by miRNAs. There is a disconnect between rigorous understanding of mechanism and the extraordinary diversity of reported roles for miRNAs. Consequences of this disconnect include confusion about the assumptions underlying the basic science of human miRNAs and slow development of therapeutics that target miRNAs. Here, we present an overview of investigations into miRNAs and their impact on gene expression. Progress in our understanding of miRNAs would be aided by a greater focus on the mechanism of miRNAs and a higher burden of evidence on researchers who seek to link expression of a particular miRNA to a biological phenotype.
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Affiliation(s)
- Audrius Kilikevicius
- Department of Pharmacology and Biochemistry, UT Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, USA
| | - Gunter Meister
- Regensburg Center for Biochemistry (RCB), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany
| | - David R Corey
- Department of Pharmacology and Biochemistry, UT Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, USA
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7
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Chu Y, Yokota S, Liu J, Kilikevicius A, Johnson KC, Corey DR. Argonaute binding within human nuclear RNA and its impact on alternative splicing. RNA (NEW YORK, N.Y.) 2021; 27:991-1003. [PMID: 34108230 PMCID: PMC8370746 DOI: 10.1261/rna.078707.121] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/09/2021] [Accepted: 06/04/2021] [Indexed: 05/03/2023]
Abstract
Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.
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Affiliation(s)
- Yongjun Chu
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, Texas 75205, USA
| | - Shinnichi Yokota
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, Texas 75205, USA
| | - Jing Liu
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, Texas 75205, USA
| | - Audrius Kilikevicius
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, Texas 75205, USA
| | - Krystal C Johnson
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, Texas 75205, USA
| | - David R Corey
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, Texas 75205, USA
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8
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Mancilla-Galindo J, Galindo-Sevilla N. Exploring the rationale for thermotherapy in COVID-19. Int J Hyperthermia 2021; 38:202-212. [PMID: 33682604 DOI: 10.1080/02656736.2021.1883127] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Increased transmissibility of the pandemic severe acute respiratory coronavirus 2 (SARS-CoV-2) has been noted to occur at lower ambient temperatures. This is seemingly related to a better replication of most respiratory viruses, including SARS-CoV-2, at lower-than-core body temperatures (i.e., 33 °C vs 37 °C). Also, intrinsic characteristics of SARS-CoV-2 make it a heat-susceptible pathogen. Thermotherapy has successfully been used to combat viral infections in plants which could otherwise result in great economic losses; 90% of viruses causing infections in plants are positive-sense single-stranded ribonucleic acid (+ssRNA) viruses, a characteristic shared by SARS-CoV-2. Thus, it is possible to envision the use of heat-based interventions (thermotherapy or mild-temperature hyperthermia) in patients with COVID-19 for which moderate cycles (every 8-12 h) of mild-temperature hyperthermia (1-2 h) have been proposed. However, there are potential safety and mechanistic concerns which could limit the use of thermotherapy only to patients with mild-to-moderate COVID-19 to prevent disease progression rather than to treat patients who have already progressed to severe-to-critical COVID-19. Here, we review the characteristics of SARS-CoV-2 which make it a heat-susceptible virus, potential host mechanisms which could be enhanced at higher temperatures to aid viral clearance, and how thermotherapy could be investigated as a modality of treatment in patients with COVID-19 while taking into consideration potential risks.
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Affiliation(s)
- Javier Mancilla-Galindo
- Facultad de Medicina, División de Investigación, Unidad de Investigación UNAM-INC, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
| | - Norma Galindo-Sevilla
- Departamento de Infectología e Inmunología, Instituto Nacional de Perinatología, Mexico City, Mexico
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9
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Perez-Mendez M, Zárate-Segura P, Salas-Benito J, Bastida-González F. siRNA Design to Silence the 3'UTR Region of Zika Virus. BIOMED RESEARCH INTERNATIONAL 2020; 2020:6759346. [PMID: 32802864 PMCID: PMC7421096 DOI: 10.1155/2020/6759346] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/15/2020] [Revised: 05/16/2020] [Accepted: 07/02/2020] [Indexed: 12/25/2022]
Abstract
The disease caused by the Zika virus (ZIKV) has positioned itself as one of the main public health problems in Mexico. One of the main reasons is it causes microcephaly and other birth defects. The transmission of ZIKV is through Aedes aegypti and Ae. albopictus mosquitoes, which are found in a larger space of the national territory. In addition, it can also be transmitted via blood transfusion, sexual relations, and maternal-fetal route. So far, there are no vaccines or specific treatments to deal with this infection. Currently, some new therapeutics such as small interfering RNAs (siRNAs) are able to regulate or suppress transcription in viruses. Therefore, in this project, an in silico siRNA was designed for the 3'UTR region of ZIKV via bioinformatics tools. The designed siRNA was synthesized and transfected into the C6/36 cell line, previously infected with ZIKV in order to assess the ability of the siRNA to inhibit viral replication. The designed siRNA was able to inhibit significantly (p < 0.05) ZIKV replication; this siRNA could be considered a potential therapeutic towards the disease that causes ZIKV and the medical problems generated.
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Affiliation(s)
- María Perez-Mendez
- Laboratorio de Medicina Traslacional, Escuela Superior de Medicina, Instituto Politécnico Nacional, St. Salvador Díaz Mirón Esquina Plan de San Luis, Santo Tomas, Miguel Hidalgo, CDMX 11340, Mexico
| | - Paola Zárate-Segura
- Laboratorio de Medicina Traslacional, Escuela Superior de Medicina, Instituto Politécnico Nacional, St. Salvador Díaz Mirón Esquina Plan de San Luis, Santo Tomas, Miguel Hidalgo, CDMX 11340, Mexico
| | - Juan Salas-Benito
- Laboratorio de Biomedicina Molecular 3, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Guillermo Massieu Helguera 239, La Escalera-Ticomán, Gustavo A. Madero, CDMX 07329, Mexico
| | - Fernando Bastida-González
- Laboratorio de Biología Molecular, Laboratorio Estatal de Salud Pública del Estado de México, Paseo Tollocan s/n, La Moderna de la Cruz, EDOMEX, Toluca, 50180, Mexico
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10
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Chu Y, Kilikevicius A, Liu J, Johnson KC, Yokota S, Corey DR. Argonaute binding within 3'-untranslated regions poorly predicts gene repression. Nucleic Acids Res 2020; 48:7439-7453. [PMID: 32501500 PMCID: PMC7367155 DOI: 10.1093/nar/gkaa478] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2020] [Revised: 05/14/2020] [Accepted: 05/26/2020] [Indexed: 02/07/2023] Open
Abstract
Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3'-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3'-UTR. Our results suggest that assumptions about miRNA action should be re-examined.
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Affiliation(s)
- Yongjun Chu
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, TX 75205, USA
| | - Audrius Kilikevicius
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, TX 75205, USA
| | - Jing Liu
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, TX 75205, USA
| | - Krystal C Johnson
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, TX 75205, USA
| | - Shinnichi Yokota
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, TX 75205, USA
| | - David R Corey
- UT Southwestern Medical Center, Departments of Pharmacology and Biochemistry, Dallas, TX 75205, USA
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11
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Adiliaghdam F, Basavappa M, Saunders TL, Harjanto D, Prior JT, Cronkite DA, Papavasiliou N, Jeffrey KL. A Requirement for Argonaute 4 in Mammalian Antiviral Defense. Cell Rep 2020; 30:1690-1701.e4. [PMID: 32049003 PMCID: PMC7039342 DOI: 10.1016/j.celrep.2020.01.021] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Revised: 09/09/2019] [Accepted: 01/06/2020] [Indexed: 01/07/2023] Open
Abstract
While interferon (IFN) responses are critical for mammalian antiviral defense, induction of antiviral RNA interference (RNAi) is evident. To date, individual functions of the mammalian RNAi and micro RNA (miRNA) effector proteins Argonautes 1-4 (AGO1-AGO4) during virus infection remain undetermined. AGO2 was recently implicated in mammalian antiviral defense, so we examined antiviral activity of AGO1, AGO3, or AGO4 in IFN-competent immune cells. Only AGO4-deficient cells are hyper-susceptible to virus infection. AGO4 antiviral function is both IFN dependent and IFN independent, since AGO4 promotes IFN but also maintains antiviral capacity following prevention of IFN signaling or production. We identified AGO-loaded virus-derived short interfering RNAs (vsiRNAs), a molecular marker of antiviral RNAi, in macrophages infected with influenza or influenza lacking the IFN and RNAi suppressor NS1, which are uniquely diminished without AGO4. Importantly, AGO4-deficient influenza-infected mice have significantly higher burden and viral titers in vivo. Together, our data assign an essential role for AGO4 in mammalian antiviral defense.
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Affiliation(s)
- Fatemeh Adiliaghdam
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Megha Basavappa
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Tahnee L Saunders
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Dewi Harjanto
- Division of Immune Diversity, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - John T Prior
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - D Alexander Cronkite
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Nina Papavasiliou
- Division of Immune Diversity, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Kate L Jeffrey
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
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12
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Linck-Paulus L, Hellerbrand C, Bosserhoff AK, Dietrich P. Dissimilar Appearances Are Deceptive-Common microRNAs and Therapeutic Strategies in Liver Cancer and Melanoma. Cells 2020; 9:E114. [PMID: 31906510 PMCID: PMC7017070 DOI: 10.3390/cells9010114] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Revised: 12/20/2019] [Accepted: 12/23/2019] [Indexed: 12/13/2022] Open
Abstract
: In this review, we summarize the current knowledge on miRNAs as therapeutic targets in two cancer types that were frequently described to be driven by miRNAs-melanoma and hepatocellular carcinoma (HCC). By focusing on common microRNAs and associated pathways in these-at first sight-dissimilar cancer types, we aim at revealing similar molecular mechanisms that are evolved in microRNA-biology to drive cancer progression. Thereby, we also want to outlay potential novel therapeutic strategies. After providing a brief introduction to general miRNA biology and basic information about HCC and melanoma, this review depicts prominent examples of potent oncomiRs and tumor-suppressor miRNAs, which have been proven to drive diverse cancer types including melanoma and HCC. To develop and apply miRNA-based therapeutics for cancer treatment in the future, it is essential to understand how miRNA dysregulation evolves during malignant transformation. Therefore, we highlight important aspects such as genetic alterations, miRNA editing and transcriptional regulation based on concrete examples. Furthermore, we expand our illustration by focusing on miRNA-associated proteins as well as other regulators of miRNAs which could also provide therapeutic targets. Finally, design and delivery strategies of miRNA-associated therapeutic agents as well as potential drawbacks are discussed to address the question of how miRNAs might contribute to cancer therapy in the future.
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Affiliation(s)
- Lisa Linck-Paulus
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany; (L.L.-P.); (C.H.)
| | - Claus Hellerbrand
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany; (L.L.-P.); (C.H.)
- Comprehensive Cancer Center (CCC) Erlangen-EMN, 91054 Erlangen, Germany
| | - Anja K. Bosserhoff
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany; (L.L.-P.); (C.H.)
- Comprehensive Cancer Center (CCC) Erlangen-EMN, 91054 Erlangen, Germany
| | - Peter Dietrich
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany; (L.L.-P.); (C.H.)
- Department of Medicine 1, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany
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13
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Sala L, Chandrasekhar S, Vidigal JA. AGO unchained: Canonical and non-canonical roles of Argonaute proteins in mammals. Front Biosci (Landmark Ed) 2020; 25:1-42. [PMID: 31585876 DOI: 10.2741/4793] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Argonaute (AGO) proteins play key roles in animal physiology by binding to small RNAs and regulating the expression of their targets. In mammals, they do so through two distinct pathways: the miRNA pathway represses genes through a multiprotein complex that promotes both decay and translational repression; the siRNA pathway represses transcripts through direct Ago2-mediated cleavage. Here, we review our current knowledge of mechanistic details and physiological requirements of both these pathways and briefly discuss their implications to human disease.
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Affiliation(s)
- Laura Sala
- Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA
| | - Srividya Chandrasekhar
- Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA
| | - Joana A Vidigal
- Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA,
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14
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Abstract
Small RNAs govern almost every biological process in eukaryotes associating with the Argonaute (AGO) proteins to form the RNA-induced silencing complex (mRISC). AGO proteins constitute the core of RISCs with different members having variety of protein-binding partners and biochemical properties. This review focuses on the AGO subfamily of the AGOs that are ubiquitously expressed and are associated with small RNAs. The structure, function and role of the AGO proteins in the cell is discussed in detail.
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Affiliation(s)
- Saife Niaz
- Department of Biotechnology, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India
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15
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Laudadio I, Orso F, Azzalin G, Calabrò C, Berardinelli F, Coluzzi E, Gioiosa S, Taverna D, Sgura A, Carissimi C, Fulci V. AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC. EMBO Rep 2018; 20:embr.201845969. [PMID: 30591524 DOI: 10.15252/embr.201845969] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Revised: 11/16/2018] [Accepted: 11/26/2018] [Indexed: 12/31/2022] Open
Abstract
Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) constitute the core telomerase enzyme that maintains the length of telomeres. Telomere maintenance is affected in a broad range of cancer and degenerative disorders. Taking advantage of gain- and loss-of-function approaches, we show that Argonaute 2 (AGO2) promotes telomerase activity and stimulates the association between TERT and TERC AGO2 depletion results in shorter telomeres as well as in lower proliferation rates in vitro and in vivo We also demonstrate that AGO2 interacts with TERC and with a newly identified sRNA (terc-sRNA), arising from the H/ACA box of TERC Notably, terc-sRNA is sufficient to enhance telomerase activity when overexpressed. Analyses of sRNA-Seq datasets show that terc-sRNA is detected in primary human tissues and increases in tumors as compared to control tissues. Collectively, these data uncover a new layer of complexity in the regulation of telomerase activity by AGO2 and might lay the foundation for new therapeutic targets in tumors and telomere diseases.
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Affiliation(s)
- Ilaria Laudadio
- Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy
| | - Francesca Orso
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy
| | - Gianluca Azzalin
- Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy
| | - Carlo Calabrò
- Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy
| | | | - Elisa Coluzzi
- Department of Science, University of Rome "Roma Tre", Rome, Italy
| | - Silvia Gioiosa
- CNR, Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari (IBIOM), Bari, Italy
| | - Daniela Taverna
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy
| | - Antonella Sgura
- Department of Science, University of Rome "Roma Tre", Rome, Italy
| | - Claudia Carissimi
- Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy
| | - Valerio Fulci
- Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy
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16
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Bridge KS, Shah KM, Li Y, Foxler DE, Wong SCK, Miller DC, Davidson KM, Foster JG, Rose R, Hodgkinson MR, Ribeiro PS, Aboobaker AA, Yashiro K, Wang X, Graves PR, Plevin MJ, Lagos D, Sharp TV. Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins. Cell Rep 2018; 20:173-187. [PMID: 28683311 PMCID: PMC5507773 DOI: 10.1016/j.celrep.2017.06.027] [Citation(s) in RCA: 46] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2015] [Revised: 04/27/2017] [Accepted: 06/09/2017] [Indexed: 10/26/2022] Open
Abstract
As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.
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Affiliation(s)
- Katherine S Bridge
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Kunal M Shah
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Yigen Li
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Daniel E Foxler
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Sybil C K Wong
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Duncan C Miller
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Kathryn M Davidson
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - John G Foster
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - Ruth Rose
- School of Biological and Chemical Sciences, Queen Mary University of London, Fogg Building, Mile End Road, London E1 4NS, UK
| | | | - Paulo S Ribeiro
- Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK
| | - A Aziz Aboobaker
- Department of Zoology, University of Oxford, The Tinbergen Building, South Parks Road, Oxford OX1 3PS, UK
| | - Kenta Yashiro
- Cardiac Regeneration and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Xiaozhong Wang
- Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA
| | - Paul R Graves
- Department of Radiation Oncology, New York-Presbyterian Brooklyn Methodist Hospital, 506 6th Street, Brooklyn, NY 11215, USA
| | - Michael J Plevin
- Department of Biology, University of York, Heslington, York YO10 5DD, UK
| | - Dimitris Lagos
- Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, Heslington, York YO10 5DD, UK
| | - Tyson V Sharp
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK.
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17
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Herrera-Carrillo E, Berkhout B. Dicer-independent processing of small RNA duplexes: mechanistic insights and applications. Nucleic Acids Res 2017; 45:10369-10379. [PMID: 28977573 PMCID: PMC5737282 DOI: 10.1093/nar/gkx779] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Accepted: 08/24/2017] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs (miRNAs) play a pivotal role in the regulation of cellular gene expression via the conserved RNA interference (RNAi) mechanism. Biogenesis of the unusual miR-451 does not require Dicer. This molecule is instead processed by the Argonaute 2 (Ago2) enzyme. Similarly, unconventional short hairpin RNA (shRNA) molecules have been designed as miR-451 mimics that rely exclusively on Ago2 for maturation. We will review recent progress made in the understanding of this alternative processing route. Next, we describe different Dicer-independent shRNA designs that have been developed and discuss their therapeutic advantages and disadvantages. As an example, we will present the route towards development of a durable gene therapy against HIV-1.
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Affiliation(s)
- Elena Herrera-Carrillo
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, the Netherlands
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, the Netherlands
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18
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Harwig A, Kruize Z, Yang Z, Restle T, Berkhout B. Analysis of AgoshRNA maturation and loading into Ago2. PLoS One 2017; 12:e0183269. [PMID: 28809941 PMCID: PMC5557517 DOI: 10.1371/journal.pone.0183269] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2017] [Accepted: 08/01/2017] [Indexed: 12/11/2022] Open
Abstract
The RNA interference (RNAi) pathway was recently expanded by the discovery of multiple alternative pathways for processing of natural microRNA (miRNA) and man-made short hairpin RNA (shRNA) molecules. One non-canonical pathway bypasses Dicer cleavage and requires instead processing by Argonaute2 (Ago2), which also executes the subsequent silencing step. We named these molecules AgoshRNA, which generate only a single active RNA strand and thus avoid off-target effects that can be induced by the passenger strand of a regular shRNA. Previously, we characterized AgoshRNA processing by deep sequencing and demonstrated that—after Ago2 cleavage—AgoshRNAs acquire a short 3’ tail of 1–3 A-nucleotides and are subsequently trimmed, likely by the poly(A)-specific ribonuclease (PARN). As a result, the mature single-stranded AgoshRNA may dock more stably into Ago2. Here we set out to analyze the activity of different synthetic AgoshRNA processing intermediates. Ago2 was found to bind preferentially to partially single-stranded AgoshRNA in vitro. In contrast, only the double-stranded AgoshRNA precursor associated with Ago2 in cells, correlating with efficient intracellular processing and reporter knockdown activity. These results suggest the presence of a cellular co-factor involved in AgoshRNA loading into Ago2 in vivo. We also demonstrate specific AgoshRNA loading in Ago2, but not Ago1/3/4, thus further reducing unwanted side effects.
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Affiliation(s)
- Alex Harwig
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Zita Kruize
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Zhenhuang Yang
- Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, Germany
| | - Tobias Restle
- Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, Germany
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
- * E-mail:
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19
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Paces J, Nic M, Novotny T, Svoboda P. Literature review of baseline information to support the risk assessment of RNAi‐based GM plants. ACTA ACUST UNITED AC 2017. [PMCID: PMC7163844 DOI: 10.2903/sp.efsa.2017.en-1246] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Jan Paces
- Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (IMG)
| | | | | | - Petr Svoboda
- Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (IMG)
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20
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Herrera-Carrillo E, Harwig A, Berkhout B. Silencing of HIV-1 by AgoshRNA molecules. Gene Ther 2017; 24:453-461. [DOI: 10.1038/gt.2017.44] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Revised: 04/13/2017] [Accepted: 05/12/2017] [Indexed: 12/17/2022]
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21
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Gerresheim GK, Dünnes N, Nieder-Röhrmann A, Shalamova LA, Fricke M, Hofacker I, Höner Zu Siederdissen C, Marz M, Niepmann M. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure. Cell Mol Life Sci 2017; 74:747-760. [PMID: 27677491 PMCID: PMC11107659 DOI: 10.1007/s00018-016-2377-9] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Revised: 08/29/2016] [Accepted: 09/21/2016] [Indexed: 02/08/2023]
Abstract
We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.
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Affiliation(s)
- Gesche K Gerresheim
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Nadia Dünnes
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Anika Nieder-Röhrmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Lyudmila A Shalamova
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
| | - Markus Fricke
- Faculty of Mathematics and Computer Science, Friedrich-Schiller-University, 07743, Jena, Germany
| | - Ivo Hofacker
- Institute for Theoretical Chemistry, University of Vienna, 1090, Vienna, Austria
| | - Christian Höner Zu Siederdissen
- Institute for Theoretical Chemistry, University of Vienna, 1090, Vienna, Austria
- Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, Universität Leipzig, 04107, Leipzig, Germany
| | - Manja Marz
- Faculty of Mathematics and Computer Science, Friedrich-Schiller-University, 07743, Jena, Germany
- FLI Leibniz Institute for Age Research, 07743, Jena, Germany
| | - Michael Niepmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany.
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22
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Völler D, Linck L, Bruckmann A, Hauptmann J, Deutzmann R, Meister G, Bosserhoff AK. Argonaute Family Protein Expression in Normal Tissue and Cancer Entities. PLoS One 2016; 11:e0161165. [PMID: 27518285 PMCID: PMC4982624 DOI: 10.1371/journal.pone.0161165] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 08/01/2016] [Indexed: 11/30/2022] Open
Abstract
The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease.
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Affiliation(s)
- Daniel Völler
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Lisa Linck
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Astrid Bruckmann
- Biochemistry Center Regensburg, University of Regensburg, Regensburg, Germany
| | - Judith Hauptmann
- Biochemistry Center Regensburg, University of Regensburg, Regensburg, Germany
| | - Rainer Deutzmann
- Biochemistry Center Regensburg, University of Regensburg, Regensburg, Germany
| | - Gunter Meister
- Biochemistry Center Regensburg, University of Regensburg, Regensburg, Germany
| | - Anja Katrin Bosserhoff
- Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
- * E-mail:
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23
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Yamasaki T, Kim EJ, Cerutti H, Ohama T. Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2016; 85:258-268. [PMID: 26686836 DOI: 10.1111/tpj.13107] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2015] [Revised: 11/27/2015] [Accepted: 12/07/2015] [Indexed: 06/05/2023]
Abstract
MicroRNAs (miRNAs) play important roles in diverse biological processes in eukaryotes, generally through degradation and/or inhibition of the translation of target mRNAs. MicroRNAs are loaded into Argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC) and used as guides to identify complementary transcripts. The distinct functions and features, such as associated small RNA classes and modes of silencing, of individual AGO paralogs have been well documented in multicellular eukaryotes. However, this aspect of miRNA function remains poorly understood in the unicellular green alga Chlamydomonas reinhardtii, which contains three AGO paralogs. In this study, we isolated AGO2 and AGO3 insertional mutants and confirmed that AGO3 is more abundantly expressed than AGO2. MicroRNA-directed target transcript cleavage and translational repression were impaired in the AGO3 mutant background, indicating that AGO3 can mediate both modes of silencing. In contrast, although the AGO2 mutant is not a null, the involvement of AGO2 in miRNA-directed silencing appears to be more limited. Our results strongly suggest that miRNA-mediated post-transcriptional gene silencing relies primarily on AGO3 in Chlamydomonas.
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Affiliation(s)
- Tomohito Yamasaki
- Department of Environmental Systems Engineering, Kochi University of Technology (KUT), 185 Miyanokuchi, Tosayamada, Kami, Kochi, 782-8502, Japan
| | - Eun-Jeong Kim
- School of Biological Science and Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA
| | - Heriberto Cerutti
- School of Biological Science and Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA
| | - Takeshi Ohama
- Department of Environmental Systems Engineering, Kochi University of Technology (KUT), 185 Miyanokuchi, Tosayamada, Kami, Kochi, 782-8502, Japan
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24
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Nishi K, Takahashi T, Suzawa M, Miyakawa T, Nagasawa T, Ming Y, Tanokura M, Ui-Tei K. Control of the localization and function of a miRNA silencing component TNRC6A by Argonaute protein. Nucleic Acids Res 2015; 43:9856-73. [PMID: 26446993 PMCID: PMC4787778 DOI: 10.1093/nar/gkv1026] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2015] [Accepted: 09/28/2015] [Indexed: 12/12/2022] Open
Abstract
GW182 family proteins play important roles in microRNA (miRNA)-mediated RNA silencing. They directly interact with Argonaute (Ago) proteins in processing bodies (P bodies), cytoplasmic foci involved in mRNA degradation and storage. Recently, we revealed that a human GW182 family protein, TNRC6A, is a nuclear-cytoplasmic shuttling protein, and its subcellular localization is regulated by its own nuclear localization signal and nuclear export signal. Regarding the further controlling mechanism of TNRC6A subcellular localization, we found that TNRC6A protein is tethered in P bodies by direct interaction with Ago2 under Ago2 overexpression condition in HeLa cells. Furthermore, it was revealed that such Ago proteins might be strongly tethered in the P bodies through Ago-bound small RNAs. Thus, our results indicate that TNRC6A subcellular localization is substantially controlled by the interaction with Ago proteins. Furthermore, it was also revealed that the TNRC6A subcellular localization affects the RNA silencing activity.
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Affiliation(s)
- Kenji Nishi
- Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
| | - Tomoko Takahashi
- Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
| | - Masataka Suzawa
- Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
| | - Takuya Miyakawa
- Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
| | - Tatsuya Nagasawa
- Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
| | - Yvelt Ming
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba-ken 277-8651, Japan
| | - Masaru Tanokura
- Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
| | - Kumiko Ui-Tei
- Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba-ken 277-8651, Japan
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25
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Abstract
During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.
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26
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Méndez C, Ahlenstiel CL, Kelleher AD. Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus. World J Virol 2015; 4:219-244. [PMID: 26279984 PMCID: PMC4534814 DOI: 10.5501/wjv.v4.i3.219] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2014] [Revised: 01/24/2015] [Accepted: 04/29/2015] [Indexed: 02/05/2023] Open
Abstract
While human immunodeficiency virus 1 (HIV-1) infection is controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference (RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by non-coding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing (TGS), as well as fine-tuning their expression through post-transcriptional gene silencing (PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.
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Schraivogel D, Schindler SG, Danner J, Kremmer E, Pfaff J, Hannus S, Depping R, Meister G. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels. Nucleic Acids Res 2015; 43:7447-61. [PMID: 26170235 PMCID: PMC4551936 DOI: 10.1093/nar/gkv705] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2015] [Accepted: 07/01/2015] [Indexed: 12/28/2022] Open
Abstract
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels.
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Affiliation(s)
- Daniel Schraivogel
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
| | - Susann G Schindler
- Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany
| | - Johannes Danner
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
| | - Elisabeth Kremmer
- Institute of Molecular Immunology, Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Marchioninistraße 25, 81377 Munich, Germany
| | - Janina Pfaff
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
| | - Stefan Hannus
- Intana Biosciences GmbH, Lochhamerstrasse 29A, 82152 Martinsried/Planegg, Germany
| | - Reinhard Depping
- Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany
| | - Gunter Meister
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
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Liu YP, Karg M, Herrera-Carrillo E, Berkhout B. Towards Antiviral shRNAs Based on the AgoshRNA Design. PLoS One 2015; 10:e0128618. [PMID: 26087209 PMCID: PMC4472832 DOI: 10.1371/journal.pone.0128618] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2015] [Accepted: 04/30/2015] [Indexed: 12/31/2022] Open
Abstract
RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these "AgoshRNA" molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.
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Affiliation(s)
- Ying Poi Liu
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, the Netherlands
| | - Margarete Karg
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, the Netherlands
| | - Elena Herrera-Carrillo
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, the Netherlands
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, the Netherlands
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Abstract
Post-transcriptional gene silencing is a widely used method to suppress gene expression. Unfortunately only a portion of siRNAs do successfully reduce gene expression. Target mRNA secondary structures and siRNA-mRNA thermodynamic features are believed to contribute to the silencing activity. However, there is still an open discussion as to what determines siRNA efficacy. In this retrospective study, we analysed the target accessibility comparing very high (VH) compared with low (L) efficacy siRNA sequences obtained from the siRecords Database. We determined the contribution of mRNA target local secondary structures on silencing efficacy. Both the univariable and the multivariable logistic regression evidenced no relationship between siRNA efficacy and mRNA target secondary structures. Moreover, none of the thermodynamic and sequence-base parameters taken into consideration (H-b index, ΔG°overall, ΔG°duplex, ΔG°break-target and GC%) was associated with siRNA efficacy. We found that features believed to be predictive of silencing efficacy are not confirmed to be so when externally evaluated in a large heterogeneous sample. Although it was proposed that silencing efficacy could be influenced by local target accessibility we show that this could be not generalizable because of the diversity of experimental setting that may not be representative of biological systems especially in view of the many local protein factors, usually not taken into consideration, which could hamper the silencing process. We analysed several siRNA-mRNA target features involved in silencing efficacy. We found out that features believed to be predictive of silencing efficacy are not such when transferred to a larger dataset of experiments and different experimental settings.
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Jahns H, Roos M, Imig J, Baumann F, Wang Y, Gilmour R, Hall J. Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs. Nat Commun 2015; 6:6317. [PMID: 25744034 PMCID: PMC4366519 DOI: 10.1038/ncomms7317] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2014] [Accepted: 01/19/2015] [Indexed: 12/18/2022] Open
Abstract
An established means of improving the pharmacokinetics properties of oligoribonucleotides (ORNs) is to exchange their phosphodiester linkages for phosphorothioates (PSs). However, this strategy has not been pursued for small interfering RNAs (siRNAs), possibly because of sporadic reports that PS siRNAs show reduced inhibitory activity. The PS group is chiral at phosphorous (Rp/Sp centres), and conventional solid-phase synthesis of PS ORNs produces a population of diastereoisomers. Here we show that the choice of the activating agent for the synthesis of a PS ORN influences the Rp/Sp ratio of PS linkages throughout the strand. Furthermore, PS siRNAs composed of ORNs with a higher fraction of Rp centres show greater resistance to nucleases in serum and are more effective inhibitors in cells than their Sp counterparts. The finding that a stereochemically biased population of ORN diastereoisomers can be synthesized and exploited pharmacologically is important because uniform PS modification of siRNAs may provide a useful compromise of their pharmacokinetics and pharmacodynamics properties in RNAi therapeutics. Therapeutic oligonucleotides can be made more stable by substituting their achiral phosphodiester groups for chiral phosphorothioate linkages. Here, the authors present a synthesis of phosphorothioated RNAs, where the activator controls strand stereochemistry, and also the activity of assembled siRNAs.
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Affiliation(s)
- Hartmut Jahns
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich, Switzerland
| | - Martina Roos
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich, Switzerland
| | - Jochen Imig
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich, Switzerland
| | - Fabienne Baumann
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich, Switzerland
| | - Yuluan Wang
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich, Switzerland
| | - Ryan Gilmour
- Institute for Organic Chemistry, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany
| | - Jonathan Hall
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich, Switzerland
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31
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Nicklas S, Okawa S, Hillje AL, González-Cano L, Del Sol A, Schwamborn JC. The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs. Nucleic Acids Res 2015; 43:2638-54. [PMID: 25722370 PMCID: PMC4357729 DOI: 10.1093/nar/gkv138] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein–protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.
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Affiliation(s)
- Sarah Nicklas
- Stem Cell Biology and Regeneration Group, Institute of Cell Biology, ZMBE, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany Developmental and Cellular Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, L-4362 Esch-Belval, Luxembourg
| | - Satoshi Okawa
- Computational Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, L-4362 Esch-Belval, Luxembourg
| | - Anna-Lena Hillje
- Stem Cell Biology and Regeneration Group, Institute of Cell Biology, ZMBE, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany Developmental and Cellular Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, L-4362 Esch-Belval, Luxembourg
| | - Laura González-Cano
- Stem Cell Biology and Regeneration Group, Institute of Cell Biology, ZMBE, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany Developmental and Cellular Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, L-4362 Esch-Belval, Luxembourg
| | - Antonio Del Sol
- Computational Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, L-4362 Esch-Belval, Luxembourg
| | - Jens C Schwamborn
- Stem Cell Biology and Regeneration Group, Institute of Cell Biology, ZMBE, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany Developmental and Cellular Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, L-4362 Esch-Belval, Luxembourg
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32
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Sun G, Yeh SY, Yuan CWY, Chiu MJY, Yung BSH, Yen Y. Molecular Properties, Functional Mechanisms, and Applications of Sliced siRNA. MOLECULAR THERAPY-NUCLEIC ACIDS 2015; 4:e221. [PMID: 25602583 PMCID: PMC4345305 DOI: 10.1038/mtna.2014.73] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/31/2014] [Accepted: 12/06/2014] [Indexed: 11/09/2022]
Abstract
Using pre-miR-451 as a model molecule, we have characterized the general molecular properties of small hairpin RNAs that are processed into potent small interfering RNAs (siRNA) by Argonaute2 (Ago2). The Ago2-sliced siRNAs (sli-siRNAs) have the same silencing potency as the classical Dicer diced siRNAs (di-siRNAs) but have dramatically reduced unwanted sense strand activities. We have built vectors with the constitutive or inducible U6 promoter that can express sli-siRNAs in mammalian cells, in which the sli-siRNAs can be correctly processed to repress target genes. As a proof of principle for potential applications of sli-siRNAs in vivo, we show that the expression of one Ago2 shRNA-1148 in HCT-116 colon cancer cells knocked down RRM2 expression and reduced the proliferation and invasiveness of the cells. The defined sli-siRNA model molecules and the expression systems established in this study will facilitate the design and application of sli-siRNAs as novel potent RNAi triggers with reduced off-target effects.
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Affiliation(s)
- Guihua Sun
- Department of Molecular Pharmacology, Beckman Research Institute of the City of Hope, Duarte, California, USA
| | - Spencer Yele Yeh
- Summer Interns, Beckman Research Institute of the City of Hope, Duarte, California, USA
| | | | | | - Bryan Shing Hei Yung
- Summer Interns, Beckman Research Institute of the City of Hope, Duarte, California, USA
| | - Yun Yen
- Department of Molecular Pharmacology, Beckman Research Institute of the City of Hope, Duarte, California, USA
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33
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Designing Ago2-specific siRNA/shRNA to Avoid Competition with Endogenous miRNAs. MOLECULAR THERAPY. NUCLEIC ACIDS 2014; 3:e176. [PMID: 25025466 PMCID: PMC4121517 DOI: 10.1038/mtna.2014.27] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/05/2014] [Accepted: 05/23/2014] [Indexed: 12/22/2022]
Abstract
Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 structure with completely complementary stem, which significantly improves the Ago2 specificity. This shRNA was shown to be Ago2-specific, and maintain target-silencing ability while avoiding competition with endogenous miRNAs by not competing for Agos 1, 3, and 4. We conclude that modified pre-miR-451 structure provides a general platform to design shRNAs that significantly reduce perturbation of miRNA function.
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34
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Dueck A, Meister G. Assembly and function of small RNA – Argonaute protein complexes. Biol Chem 2014; 395:611-29. [DOI: 10.1515/hsz-2014-0116] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2014] [Accepted: 02/28/2014] [Indexed: 01/05/2023]
Abstract
Abstract
Small RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs) or Piwi-interacting RNAs (piRNAs) are important regulators of gene expression in various organisms. Small RNAs bind to a member of the Argonaute protein family and are incorporated into larger structures that mediate diverse gene silencing events. The loading of Argonaute proteins with small RNAs is aided by a number of auxiliary factors as well as ATP hydrolysis. This review will focus on the mechanisms of Argonaute loading in different organisms. Furthermore, we highlight the versatile functions of small RNA-Argonaute protein complexes in organisms from all three kingdoms of life.
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35
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Hannus M, Beitzinger M, Engelmann JC, Weickert MT, Spang R, Hannus S, Meister G. siPools: highly complex but accurately defined siRNA pools eliminate off-target effects. Nucleic Acids Res 2014; 42:8049-61. [PMID: 24875475 PMCID: PMC4081087 DOI: 10.1093/nar/gku480] [Citation(s) in RCA: 134] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.
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Affiliation(s)
- Michael Hannus
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany Intana Biosciences GmbH, Lochhamerstrasse 29A, 82152 Martinsried/Planegg, Germany siTools Biotech GmbH, Lochhamerstrasse 29A, 82152 Martinsried/Planegg, Germany
| | - Michaela Beitzinger
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany siTools Biotech GmbH, Lochhamerstrasse 29A, 82152 Martinsried/Planegg, Germany
| | - Julia C Engelmann
- Department of Statistical Bioinformatics, Institute for Functional Genomics, University of Regensburg, Josef-Engert-Straße 9, 93053 Regensburg, Germany
| | - Marie-Theresa Weickert
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
| | - Rainer Spang
- Department of Statistical Bioinformatics, Institute for Functional Genomics, University of Regensburg, Josef-Engert-Straße 9, 93053 Regensburg, Germany
| | - Stefan Hannus
- Intana Biosciences GmbH, Lochhamerstrasse 29A, 82152 Martinsried/Planegg, Germany
| | - Gunter Meister
- Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
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36
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Kandeel M, Al-Taher A, Nakashima R, Sakaguchi T, Kandeel A, Nagaya Y, Kitamura Y, Kitade Y. Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain. PLoS One 2014; 9:e94538. [PMID: 24788663 PMCID: PMC4008379 DOI: 10.1371/journal.pone.0094538] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2013] [Accepted: 03/18/2014] [Indexed: 12/27/2022] Open
Abstract
Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3′-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3′-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.
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Affiliation(s)
- Mahmoud Kandeel
- Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine and Animal Resources, King Faisal University, Alhofuf, Alahsa, Saudi Arabia
- Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelshikh University, Kafrelshikh, Egypt
| | - Abdullah Al-Taher
- Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine and Animal Resources, King Faisal University, Alhofuf, Alahsa, Saudi Arabia
| | - Remi Nakashima
- United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan
| | - Tomoya Sakaguchi
- Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan
| | - Ali Kandeel
- Department of Biology, Faculty of Sciences and Arts, Alkamil Branch, King Abdul Aziz University, Alkamil, Saudi Arabia
- Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
| | - Yuki Nagaya
- Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan
| | - Yoshiaki Kitamura
- United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan
| | - Yukio Kitade
- United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan
- Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan
- * E-mail:
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37
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Booy EP, Howard R, Marushchak O, Ariyo EO, Meier M, Novakowski SK, Deo SR, Dzananovic E, Stetefeld J, McKenna SA. The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1. Nucleic Acids Res 2013; 42:3346-61. [PMID: 24369427 PMCID: PMC3950718 DOI: 10.1093/nar/gkt1340] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3′-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non–quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3′-untranslated region.
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Affiliation(s)
- Evan P Booy
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada, University of Manitoba, Winnipeg, Manitoba, Canada, Department of Biochemistry and Molecular Biology, University of British Columbia, V6T 1Z4 Vancouver, British Columbia, Canada and Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2
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38
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Börner K, Niopek D, Cotugno G, Kaldenbach M, Pankert T, Willemsen J, Zhang X, Schürmann N, Mockenhaupt S, Serva A, Hiet MS, Wiedtke E, Castoldi M, Starkuviene V, Erfle H, Gilbert DF, Bartenschlager R, Boutros M, Binder M, Streetz K, Kräusslich HG, Grimm D. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines. Nucleic Acids Res 2013; 41:e199. [PMID: 24049077 PMCID: PMC3834839 DOI: 10.1093/nar/gkt836] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2013] [Revised: 08/21/2013] [Accepted: 08/25/2013] [Indexed: 12/31/2022] Open
Abstract
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
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Affiliation(s)
- Kathleen Börner
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Dominik Niopek
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Gabriella Cotugno
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Michaela Kaldenbach
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Teresa Pankert
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Joschka Willemsen
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Xian Zhang
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Nina Schürmann
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Stefan Mockenhaupt
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Andrius Serva
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Marie-Sophie Hiet
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Ellen Wiedtke
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Mirco Castoldi
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Vytaute Starkuviene
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Holger Erfle
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Daniel F. Gilbert
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Michael Boutros
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Marco Binder
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Konrad Streetz
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Hans-Georg Kräusslich
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
| | - Dirk Grimm
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
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Conrad KD, Niepmann M. The role of microRNAs in hepatitis C virus RNA replication. Arch Virol 2013; 159:849-62. [PMID: 24158346 DOI: 10.1007/s00705-013-1883-4] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2013] [Accepted: 09/28/2013] [Indexed: 12/16/2022]
Abstract
Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5' end of the 5' untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA's 5' end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5'-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.
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Affiliation(s)
- K Dominik Conrad
- Institute of Biochemistry, School of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany
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40
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Abstract
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.
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Abstract
Multiple Argonaute proteins are implicated in gene silencing by RNA interference (RNAi), but only one is known to be an endonuclease that can cleave target mRNAs. Chimeric Argonaute proteins now reveal an unexpected mechanism by which mutations distal to the catalytic center can unmask intrinsic catalytic activity, results hinting at structurally mediated regulation.
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42
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Molecular dissection of human Argonaute proteins by DNA shuffling. Nat Struct Mol Biol 2013; 20:818-26. [PMID: 23748378 DOI: 10.1038/nsmb.2607] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2013] [Accepted: 05/13/2013] [Indexed: 01/01/2023]
Abstract
A paramount task in RNA interference research is to decipher the complex biology of cellular effectors, exemplified in humans by four pleiotropic Argonaute proteins (Ago1-Ago4). Here, we exploited DNA family shuffling, a molecular evolution technology, to generate chimeric Ago protein libraries for dissection of intricate phenotypes independently of prior structural knowledge. Through shuffling of human Ago2 and Ago3, we discovered two N-terminal motifs that govern RNA cleavage in concert with the PIWI domain. Structural modeling predicts an impact on protein rigidity and/or RNA-PIWI alignment, suggesting new mechanistic explanations for Ago3's slicing deficiency. Characterization of hybrids including Ago1 and Ago4 solidifies that slicing requires the juxtaposition and combined action of multiple disseminated modules. We also present a Gateway library of codon-optimized chimeras of human Ago1-Ago4 and molecular evolution analysis software as resources for future investigations into RNA interference sequence-structure-function relationships.
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Abstract
Small-RNA-guided gene regulation has emerged as one of the fundamental principles in cell function, and the major protein players in this process are members of the Argonaute protein family. Argonaute proteins are highly specialized binding modules that accommodate the small RNA component - such as microRNAs (miRNAs), short interfering RNAs (siRNAs) or PIWI-associated RNAs (piRNAs) - and coordinate downstream gene-silencing events by interacting with other protein factors. Recent work has made progress in our understanding of classical Argonaute-mediated gene-silencing principles, such as the effects on mRNA translation and decay, but has also implicated Argonaute proteins in several other cellular processes, such as transcriptional regulation and splicing.
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Hauptmann J, Dueck A, Harlander S, Pfaff J, Merkl R, Meister G. Turning catalytically inactive human Argonaute proteins into active slicer enzymes. Nat Struct Mol Biol 2013; 20:814-7. [PMID: 23665583 DOI: 10.1038/nsmb.2577] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2013] [Accepted: 04/04/2013] [Indexed: 01/15/2023]
Abstract
Argonaute proteins interact with small RNAs that guide them to complementary target RNAs, thus leading to inhibition of gene expression. Some but not all Argonaute proteins are endonucleases and can cleave the complementary target RNA. Here, we have mutated inactive human Ago1 and Ago3 and generated catalytic Argonaute proteins. We find that two short sequence elements at the N terminus are important for activity. In addition, PIWI-domain mutations in Ago1 may misarrange the catalytic center. Our work helps in understanding of the structural requirements that make an Argonaute protein an active endonucleolytic enzyme.
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Affiliation(s)
- Judith Hauptmann
- Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, Germany
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45
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Vaish N, Agarwal P. The design, selection, and evaluation of highly specific and functional siRNA incorporating unlocked nucleobase analogs. Methods Mol Biol 2013; 942:111-34. [PMID: 23027048 DOI: 10.1007/978-1-62703-119-6_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The efficient and specific silencing of genes via RNA interference (RNAi) for functional genomics and therapeutics depends on careful consideration of the factors that affect the functionality of small interfering RNA (siRNA). These factors include (1) the length of sequence available for siRNA targeting of an mRNA, (2) the structural and thermodynamic properties of target and siRNA sequences, (3) the mechanisms of siRNA off-target effects, and (4) the susceptibility of siRNA degradation when exposed to nucleases in serum and inside cells. Incorporation of Unlocked Nucleobase analogs (UNAs) in the siRNA design offers an attractive approach to design highly efficacious siRNAs with dramatically reduced off-target activity. Here, we describe methods and principles pertaining to the design, selection and screening of optimal siRNAs containing UNA.
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Conrad KD, Giering F, Erfurth C, Neumann A, Fehr C, Meister G, Niepmann M. MicroRNA-122 dependent binding of Ago2 protein to hepatitis C virus RNA is associated with enhanced RNA stability and translation stimulation. PLoS One 2013; 8:e56272. [PMID: 23405269 PMCID: PMC3566042 DOI: 10.1371/journal.pone.0056272] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2012] [Accepted: 01/08/2013] [Indexed: 01/16/2023] Open
Abstract
Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.
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Affiliation(s)
- K. Dominik Conrad
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Florian Giering
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Corinna Erfurth
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Angelina Neumann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Carmen Fehr
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
| | - Gunter Meister
- Institute of Biochemistry, Faculty of Biology and Preclinical Medicine, University of Regensburg, Regensburg, Germany
| | - Michael Niepmann
- Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
- * E-mail:
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Horn T, Boutros M. Design of RNAi reagents for invertebrate model organisms and human disease vectors. Methods Mol Biol 2013; 942:315-346. [PMID: 23027059 DOI: 10.1007/978-1-62703-119-6_17] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
RNAi has become a very versatile tool to silence gene expression in a variety of organisms, in particular when classical genetic methods are missing. However, the application of this method in functional studies has raised new challenges in order to design RNAi reagents that minimize false positives and false negatives. Because the performance of reagents cannot be validated on a genome-wide scale, improved computational methods are required that consider experimentally derived quality measures. In this chapter, we describe computational methods for the design of RNAi reagents for invertebrate model organisms and human disease vectors, such as Anopheles. We describe procedures for designing short and long double-stranded RNAs for single genes, and evaluate their predicted specificity and efficiency. Using a bioinformatics pipeline we also describe how to design a genome-wide RNAi library for Anopheles gambiae.
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Affiliation(s)
- Thomas Horn
- Department of Cell and Molecular Biology, Heidelberg University, Heidelberg, Germany
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Fahim M, Larkin PJ. Designing effective amiRNA and multimeric amiRNA against plant viruses. Methods Mol Biol 2013; 942:357-77. [PMID: 23027061 DOI: 10.1007/978-1-62703-119-6_19] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
RNA-mediated virus resistance is increasingly becoming a method of choice for antiviral defense in plants when effective natural resistance is unavailable. In this chapter we discuss the design principles of artificial micro RNA (amiRNA), in which a natural miRNA precursor gene is modified to target a different species of RNA, in particular viral RNA. In addition, we explore the advantages and effectiveness of multiple amiRNAs within one polycistronic amiRNA precursor against a virus, as illustrated with Wheat streak mosaic virus, WSMV. The judicious selection of amiRNAs, which are sequences of short length as compared to other related methodologies of RNA interference, greatly assists in avoiding unintended off-targets in the host plant. The viral sequences targeted can be genomic or replicative and should be derived from conserved regions of the published WSMV genome. In short, using published folding and miRNA selection rules and algorithms, candidate miRNA sequences are selected from conserved regions between a number of WSMV genomes, and are BLASTed against wheat TIGR ESTs. Five miRNAs are selected that are least likely to interfere with the expression of transcripts from the wheat host. Then, the natural miRNA in each of the five arms of the polycistronic rice miR395 is replaced in silico with the chosen artificial miRNAs. This artificial precursor is transformed into wheat behind a ubiquitin promoter, and its integration into transformed wheat plants is confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of this methodology using an amiRNA precursor that we have termed Fanguard. The processing of amiRNAs in transgenic leaves is verified through splinted ligation assay, and the functionality of the transgene in preventing viral replication is verified by virus bioassay. Resistance is confirmed using mechanical virus inoculation over two subsequent generations. This example demonstrates the potential of polycistronic amiRNA to achieve stable immunity to economically important viruses.
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Affiliation(s)
- Muhammad Fahim
- Lab of Plant Developmental Molecular Genetics, School of Life Science and Biotechnology, Korea University, Seoul, South Korea
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Abstract
Short interfering RNAs (siRNAs) are a major research tool that allows for knock-down of target genes via selective mRNA destruction in almost all eukaryotic organisms. siRNAs typically consist of a synthetic ∼21 nucleotide (nt) RNA-duplex where one strand is designed with perfect complementarity to the target mRNA. Although siRNAs were initially thought to be very target-specific because of their design, it turned out during the last years that all siRNAs have a more or less pronounced intrinsic off-target activity which can make the interpretation of data from siRNA experiments difficult. Here we describe essential rules for siRNA design that should be taken into account in order to obtain potent siRNAs with minimal off-target activity. In addition, we describe how to control for off-target activity in siRNA experiments.
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Affiliation(s)
- Sebastian Petri
- Laboratory for RNA Biology, Biochemistry Center Regensburg (BZR), University of Regensburg, Regensburg, Germany
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Abstract
Synthetic small interfering RNAs (siRNAs) have revolutionized functional genomics in mammalian cell cultures due to their reliability, efficiency, and ease of use. This success, however, has not fully translated into siRNA applications in vivo and in siRNA therapeutics where initial optimism has been dampened by a lack of efficient delivery strategies and reports of siRNA off-target effects and immunogenicity. Encouragingly, most aspects of siRNA behavior can be addressed by careful engineering of siRNAs incorporating beneficial chemical modifications into discrete nucleotide positions during siRNA synthesis. Here, we review the literature (Subheadings 1 -3) and provide a quick guide (Subheading 4) to how the performance of siRNA can be improved by chemical modification to suit specific applications in vitro and in vivo.
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Affiliation(s)
- Jesper B Bramsen
- Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Aarhus, Denmark.
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