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Guerra-Slompo E, Cesaro G, Guimarães B, Zanchin N. Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system. Nucleic Acids Res 2023; 51:396-419. [PMID: 36610751 PMCID: PMC9841430 DOI: 10.1093/nar/gkac1217] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 11/29/2022] [Accepted: 01/03/2023] [Indexed: 01/09/2023] Open
Abstract
Trypanosoma brucei belongs to a group of protozoans presenting fragmented large subunit rRNA. Its LSU rRNA equivalent to the 25S/28S rRNA of other eukaryotes is split into six fragments, requiring additional processing for removal of the extra spacer sequences. We have used a genetic complementation strategy to further investigate the T. brucei RRP44 nuclease in pre-rRNA maturation. TbRRP44 contains both a PIN and a RNB domain whose homologues are found in association with the exosome complex. We found that the exonucleolytic activity of the RNB domain as well as the physical presence of the PIN domain are essential for TbRRP44 function, while a catalytic site mutation in the PIN domain has no detectable effect on cell growth. A new endonucleolytic cleavage site in ITS1 was identified. In addition to the 5.8S rRNA 3'-end maturation, TbRRP44 is required for degradation of the excised 5'-ETS and for removal of part of ITS1 during maturation of the 18S rRNA 3'-end. TbRRP44 deficiency leads to accumulation of many LSU intermediate precursors, most of them not detected in control cells. TbRRP44 is also required for U3 snoRNA and spliced leader processing, indicating that TbRRP44 may have a wide role in RNA processing in T. brucei.
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Affiliation(s)
- Eloise Pavão Guerra-Slompo
- Carlos Chagas Institute, Oswaldo Cruz Foundation, FIOCRUZ, R. Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba-PR, Brazil
| | - Giovanna Cesaro
- Carlos Chagas Institute, Oswaldo Cruz Foundation, FIOCRUZ, R. Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba-PR, Brazil,Biochemistry Postgraduate Program, Federal University of Paraná, Curitiba-PR, Brazil
| | - Beatriz Gomes Guimarães
- Carlos Chagas Institute, Oswaldo Cruz Foundation, FIOCRUZ, R. Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba-PR, Brazil,Biochemistry Postgraduate Program, Federal University of Paraná, Curitiba-PR, Brazil
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2
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Nepomuceno-Mejía T, Florencio-Martínez LE, Pineda-García I, Martínez-Calvillo S. Identification of factors involved in ribosome assembly in the protozoan parasite Leishmania major. Acta Trop 2022; 228:106315. [PMID: 35041807 DOI: 10.1016/j.actatropica.2022.106315] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2021] [Revised: 01/07/2022] [Accepted: 01/13/2022] [Indexed: 01/23/2023]
Abstract
Formation of the ribosome subunits is a complex and progressive cellular process that requires a plethora of non-ribosomal transient proteins and diverse small nucleolar RNAs, which are involved from the synthesis of the precursor ribosomal RNA in the nucleolus to the final ribosome processing steps in the cytoplasm. Employing PTP-tagged Nop56 as a fishing bait to capture pre-ribosomal particles by tandem affinity purifications, mass spectrometry assays and a robust in silico analysis, here we describe tens of ribosome assembly factors involved in the synthesis of both ribosomal subunits in the human pathogen Leishmania major, where the knowledge about ribosomal biogenesis is scarce. We identified a large number of proteins that participate in most stages of ribosome biogenesis in yeast and mammals. Among them, we found several putative orthologs of factors not previously identified in L. major, such as t-Utp4, t-Utp5, Rrp7, Nop9 and Nop15. Even more interesting is the fact that we identified several novel candidates that could participate in the assembly of the atypical 60S subunit in L. major, which contains eight different rRNA species. As these proteins do not seem to have a human counterpart, they have potential as targets for novel anti-leishmanial drugs. Also, numerous proteins whose function is not apparently linked to ribosome assembly were copurified, suggesting that the L. major nucleolus is a multifunctional nuclear body.
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3
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The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei. Sci Rep 2021; 11:18325. [PMID: 34526538 PMCID: PMC8443567 DOI: 10.1038/s41598-021-97020-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Accepted: 07/30/2021] [Indexed: 01/23/2023] Open
Abstract
The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.
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4
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Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing. Nat Commun 2021; 12:5268. [PMID: 34489460 PMCID: PMC8421343 DOI: 10.1038/s41467-021-25607-2] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 08/17/2021] [Indexed: 02/07/2023] Open
Abstract
Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative 'slender' to transmissible 'stumpy' bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.
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Bajak K, Leiss K, Clayton C, Erben E. A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei. J Biol Chem 2020; 295:14291-14304. [PMID: 32763974 DOI: 10.1074/jbc.ra120.014346] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Revised: 07/29/2020] [Indexed: 11/06/2022] Open
Abstract
In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.
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Affiliation(s)
- Kathrin Bajak
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany.,Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
| | - Kevin Leiss
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany
| | - Christine Clayton
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany
| | - Esteban Erben
- Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
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6
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Ribosome Profiling in Trypanosomatids. Methods Mol Biol 2019. [PMID: 30980300 DOI: 10.1007/978-1-4939-9210-2_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
Ribosomes are the machinery responsible for reading mRNAs and translating them into proteins. The ribosome profiling approach is based on high-throughput sequencing of ribosome-protected mRNAs. RNAs not harboring ribosomes are removed by nuclease digestion leaving the so-called ribosome "footprints." The purified "footprint" RNA molecules are processed into DNA libraries and their individual abundance is determined by deep sequencing. Ribosome profiling reveals the portion of transcripts which are actually protein-coding and can be used for differential gene expression analysis addressing rates of protein synthesis, and translational control and efficiency.
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Martínez-Calvillo S, Florencio-Martínez LE, Nepomuceno-Mejía T. Nucleolar Structure and Function in Trypanosomatid Protozoa. Cells 2019; 8:cells8050421. [PMID: 31071985 PMCID: PMC6562600 DOI: 10.3390/cells8050421] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2019] [Revised: 04/26/2019] [Accepted: 04/28/2019] [Indexed: 12/12/2022] Open
Abstract
The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed.
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Affiliation(s)
- Santiago Martínez-Calvillo
- Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Col. Los Reyes Iztacala, Tlalnepantla CP 54090, Estado de México, Mexico.
| | - Luis E Florencio-Martínez
- Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Col. Los Reyes Iztacala, Tlalnepantla CP 54090, Estado de México, Mexico.
| | - Tomás Nepomuceno-Mejía
- Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Col. Los Reyes Iztacala, Tlalnepantla CP 54090, Estado de México, Mexico.
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8
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Chikne V, Shanmugha Rajan K, Shalev-Benami M, Decker K, Cohen-Chalamish S, Madmoni H, Biswas VK, Kumar Gupta S, Doniger T, Unger R, Tschudi C, Ullu E, Michaeli S. Small nucleolar RNAs controlling rRNA processing in Trypanosoma brucei. Nucleic Acids Res 2019; 47:2609-2629. [PMID: 30605535 PMCID: PMC6411936 DOI: 10.1093/nar/gky1287] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Revised: 12/04/2018] [Accepted: 12/20/2018] [Indexed: 12/19/2022] Open
Abstract
In trypanosomes, in contrast to most eukaryotes, the large subunit (LSU) ribosomal RNA is fragmented into two large and four small ribosomal RNAs (srRNAs) pieces, and this additional processing likely requires trypanosome-specific factors. Here, we examined the role of 10 abundant small nucleolar RNAs (snoRNAs) involved in rRNA processing. We show that each snoRNA involved in LSU processing associates with factors engaged in either early or late biogenesis steps. Five of these snoRNAs interact with the intervening sequences of rRNA precursor, whereas the others only guide rRNA modifications. The function of the snoRNAs was explored by silencing snoRNAs. The data suggest that the LSU rRNA processing events do not correspond to the order of rRNA transcription, and that srRNAs 2, 4 and 6 which are part of LSU are processed before srRNA1. Interestingly, the 6 snoRNAs that affect srRNA1 processing guide modifications on rRNA positions that span locations from the protein exit tunnel to the srRNA1, suggesting that these modifications may serve as check-points preceding the liberation of srRNA1. This study identifies the highest number of snoRNAs so far described that are involved in rRNA processing and/or rRNA folding and highlights their function in the unique trypanosome rRNA maturation events.
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Affiliation(s)
- Vaibhav Chikne
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - K Shanmugha Rajan
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Moran Shalev-Benami
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Kathryn Decker
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Smadar Cohen-Chalamish
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Hava Madmoni
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Viplov K Biswas
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Sachin Kumar Gupta
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Tirza Doniger
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Ron Unger
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
| | - Christian Tschudi
- Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06536, USA
| | - Elisabetta Ullu
- Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06536, USA
| | - Shulamit Michaeli
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900 Israel
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Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response. mBio 2018; 9:mBio.02430-18. [PMID: 30563898 PMCID: PMC6299225 DOI: 10.1128/mbio.02430-18] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking TbPRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in TbPRMT1-depleted cells, and a large number of TbPRMT1-interacting proteins, thereby highlighting potential TbPRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with TbPRMT1, prompting analysis of the response of TbPRMT1-depleted cells to nutrient deprivation. Indeed, depletion of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that TbPRMT1 itself binds nucleic acids in vitro and in vivo, a feature completely novel to protein arginine methyltransferases.IMPORTANCE Trypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), TbPRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation.
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10
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Romaniuk MA, Cervini G, Cassola A. Regulation of RNA binding proteins in trypanosomatid protozoan parasites. World J Biol Chem 2016; 7:146-157. [PMID: 26981203 PMCID: PMC4768119 DOI: 10.4331/wjbc.v7.i1.146] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/04/2015] [Accepted: 01/29/2016] [Indexed: 02/05/2023] Open
Abstract
Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.
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11
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A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei. Sci Rep 2016; 6:21438. [PMID: 26888608 PMCID: PMC4757839 DOI: 10.1038/srep21438] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Accepted: 01/18/2016] [Indexed: 02/06/2023] Open
Abstract
Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells.
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12
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Bühlmann M, Walrad P, Rico E, Ivens A, Capewell P, Naguleswaran A, Roditi I, Matthews KR. NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway. Nucleic Acids Res 2015; 43:4491-504. [PMID: 25873624 PMCID: PMC4482084 DOI: 10.1093/nar/gkv330] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2015] [Revised: 03/30/2015] [Accepted: 03/31/2015] [Indexed: 02/03/2023] Open
Abstract
Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5'UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export.
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Affiliation(s)
- Melanie Bühlmann
- Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
| | - Pegine Walrad
- Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK Centre for Immunology and Infection, Department of Biology, University of York, YO10 5DD, UK
| | - Eva Rico
- Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
| | - Alasdair Ivens
- Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
| | - Paul Capewell
- Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
| | | | - Isabel Roditi
- Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
| | - Keith R Matthews
- Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
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13
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Kim YI, Bandyopadhyay J, Cho I, Lee J, Park DH, Cho JH. Nucleolar GTPase NOG-1 regulates development, fat storage, and longevity through insulin/IGF signaling in C. elegans. Mol Cells 2014; 37:51-7. [PMID: 24552710 PMCID: PMC3907010 DOI: 10.14348/molcells.2014.2251] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2013] [Revised: 11/28/2013] [Accepted: 11/29/2013] [Indexed: 12/11/2022] Open
Abstract
NOG1 is a nucleolar GTPase that is critical for 60S ribosome biogenesis. Recently, NOG1 was identified as one of the downstream regulators of target of rapamycin (TOR) in yeast. It is reported that TOR is involved in regulating lifespan and fat storage in Caenorhabditis elegans. Here, we show that the nog1 ortholog (T07A9.9: nog-1) in C. elegans regulates growth, development, lifespan, and fat metabolism. A green fluorescence protein (GFP) promoter assay revealed ubiquitous expression of C. elegans nog-1 from the early embryonic to the adult stage. Furthermore, the GFP-tagged NOG-1 protein is localized to the nucleus, whereas the aberrant NOG-1 protein is concentrated in the nucleolus. Functional studies of NOG-1 in C. elegans further revealed that nog-1 knockdown resulted in smaller broodsize, slower growth, increased life span, and more fat storage. Moreover, nog-1 over-expression resulted in decreased life span. Taken together, our data suggest that nog-1 in C. elegans may be an important player in regulating life span and fat storage via the insulin/IGF pathway.
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Affiliation(s)
- Young-Il Kim
- Biomedical Research Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701,
Korea
| | - Jaya Bandyopadhyay
- Department of Biotechnology, West Bengal University of Technology, Salt Lake City, Kolkata 700-064,
India
| | - Injeong Cho
- Department of Biology Education, College of Education, Chosun University, Gwangju 501-759,
Korea
| | - Juyeon Lee
- Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712,
Korea
| | - Dae Ho Park
- Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712,
Korea
| | - Jeong Hoon Cho
- Department of Biology Education, College of Education, Chosun University, Gwangju 501-759,
Korea
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14
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Sakyiama J, Zimmer SL, Ciganda M, Williams N, Read LK. Ribosome biogenesis requires a highly diverged XRN family 5'->3' exoribonuclease for rRNA processing in Trypanosoma brucei. RNA (NEW YORK, N.Y.) 2013; 19:1419-1431. [PMID: 23974437 PMCID: PMC3854532 DOI: 10.1261/rna.038547.113] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/28/2013] [Accepted: 07/10/2013] [Indexed: 05/30/2023]
Abstract
Although biogenesis of ribosomes is a crucial process in all organisms and is thus well conserved, Trypanosoma brucei ribosome biogenesis, of which maturation of rRNAs is an early step, has multiple points of divergence. Our aim was to determine whether in the processing of the pre-rRNA precursor molecule, 5'→3' exoribonuclease activity in addition to endonucleolytic cleavage is necessary in T. brucei as in other organisms. Our approach initiated with the bioinformatic identification of a putative 5'→3' exoribonuclease, XRNE, which is highly diverged from the XRN2/Rat1 enzyme responsible for rRNA processing in other organisms. Tagging this protein in vivo allowed us to classify XRNE as nucleolar by indirect immunofluorescence and identify by copurification interacting proteins, many of which were ribosomal proteins, ribosome biogenesis proteins, and/or RNA processing proteins. To determine whether XRNE plays a role in ribosome biogenesis in procyclic form cells, we inducibly depleted the protein by RNA interference. This resulted in the generation of aberrant preprocessed 18S rRNA and 5' extended 5.8S rRNA, implicating XRNE in rRNA processing. Polysome profiles of XRNE-depleted cells demonstrated abnormal features including an increase in ribosome small subunit abundance, a decrease in large subunit abundance, and defects in polysome assembly. Furthermore, the 5' extended 5.8S rRNA in XRNE-depleted cells was observed in the large subunit, monosomes, and polysomes in this gradient. Therefore, the function of XRNE in rRNA processing, presumably due to exonucleolytic activity very early in ribosome biogenesis, has consequences that persist throughout all biogenesis stages.
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MESH Headings
- Amino Acid Sequence
- Blotting, Northern
- Blotting, Western
- Cell Nucleolus
- Cells, Cultured
- Exoribonucleases/genetics
- Exoribonucleases/metabolism
- Immunoprecipitation
- Molecular Sequence Data
- Organelle Biogenesis
- Polyribosomes/genetics
- Polyribosomes/metabolism
- RNA Processing, Post-Transcriptional
- RNA, Messenger/genetics
- RNA, Ribosomal, 18S/genetics
- RNA, Ribosomal, 18S/metabolism
- Real-Time Polymerase Chain Reaction
- Reverse Transcriptase Polymerase Chain Reaction
- Ribosomal Proteins/genetics
- Ribosomal Proteins/metabolism
- Ribosomes/metabolism
- Sequence Homology, Amino Acid
- Tandem Mass Spectrometry
- Trypanosoma brucei brucei/enzymology
- Trypanosoma brucei brucei/genetics
- Trypanosoma brucei brucei/growth & development
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Affiliation(s)
- Joseph Sakyiama
- Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA
| | - Sara L. Zimmer
- Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA
| | - Martin Ciganda
- Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA
| | - Noreen Williams
- Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA
| | - Laurie K. Read
- Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA
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15
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Clayton C, Michaeli S. 3' processing in protists. WILEY INTERDISCIPLINARY REVIEWS-RNA 2010; 2:247-55. [PMID: 21957009 DOI: 10.1002/wrna.49] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Molecular biologists have traditionally focused on the very small corner of eukaryotic evolution that includes yeast and animals; even plants have been neglected. In this article, we describe the scant information that is available concerning RNA processing in the other four major eukaryotic groups, especially pathogenic protists. We focus mainly on polyadenylation and nuclear processing of stable RNAs. These processes have--where examined--been shown to be conserved, but there are many novel details. We also briefly mention other processing reactions such as splicing.
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Affiliation(s)
- Christine Clayton
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg, Germany.
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16
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A novel protein kinase localized to lipid droplets is required for droplet biogenesis in trypanosomes. EUKARYOTIC CELL 2010; 9:1702-10. [PMID: 20833891 DOI: 10.1128/ec.00106-10] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.
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17
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Chou S, Jensen BC, Parsons M, Alber T, Grundner C. The Trypanosoma brucei life cycle switch TbPTP1 is structurally conserved and dephosphorylates the nucleolar protein NOPP44/46. J Biol Chem 2010; 285:22075-81. [PMID: 20444707 PMCID: PMC2903352 DOI: 10.1074/jbc.m110.108860] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2010] [Revised: 04/19/2010] [Indexed: 12/18/2022] Open
Abstract
Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.
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Affiliation(s)
- Seemay Chou
- From the Department of Molecular and Cell Biology and QB3 Institute, University of California, Berkeley, California 94720-3200
| | - Bryan C. Jensen
- the Seattle Biomedical Research Institute, Seattle, Washington 98109-5219, and
| | - Marilyn Parsons
- the Seattle Biomedical Research Institute, Seattle, Washington 98109-5219, and
- the Department of Global Health, University of Washington, Seattle, Washington 98195-5065
| | - Tom Alber
- From the Department of Molecular and Cell Biology and QB3 Institute, University of California, Berkeley, California 94720-3200
| | - Christoph Grundner
- the Seattle Biomedical Research Institute, Seattle, Washington 98109-5219, and
- the Department of Global Health, University of Washington, Seattle, Washington 98195-5065
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18
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Worthen C, Jensen BC, Parsons M. Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei. PLoS Negl Trop Dis 2010; 4:e678. [PMID: 20454560 PMCID: PMC2864271 DOI: 10.1371/journal.pntd.0000678] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2010] [Accepted: 03/23/2010] [Indexed: 01/01/2023] Open
Abstract
Background The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage. Methodology/Principal Findings We therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D2, quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds. Conclusions/Significance Parasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways. The parasite Trypanosoma brucei causes human African trypanosomiasis, which is fatal unless treated. Currently used drugs are toxic, difficult to administer, and often are no longer effective due to drug resistance. The search for new drugs is long and expensive, and determining which compounds are worth pursuing is a key challenge in that process. In this study we sought to determine whether different compounds elicited different responses in the mammalian-infective stage of the parasite. We also examined whether genetic knockdown of parasite molecules led to similar responses. Our results show that, depending on the treatment, the replication of the parasite genomes, proper division of the cell, and mitochondrial function can be affected. Surprisingly, these different responses were not able to predict which compounds affected the long term proliferative potential of T. brucei. We found that some of the compounds had irreversible effects on the parasites within one day, so that even cells that appeared healthy could not proliferate. We suggest that determining which compounds set the parasites on a one-way journey to death may provide a means of identifying those that could lead to drugs with high efficacy.
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Affiliation(s)
- Christal Worthen
- Seattle Biomedical Research Institute, Seattle, Washington, United States of America
| | - Bryan C. Jensen
- Seattle Biomedical Research Institute, Seattle, Washington, United States of America
| | - Marilyn Parsons
- Seattle Biomedical Research Institute, Seattle, Washington, United States of America
- Department of Global Health, University of Washington, Seattle, Washington, United States of America
- * E-mail:
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19
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Identification and specific localization of tyrosine-phosphorylated proteins in Trypanosoma brucei. EUKARYOTIC CELL 2009; 8:617-26. [PMID: 19181871 PMCID: PMC2669198 DOI: 10.1128/ec.00366-08] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.
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20
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Prohaska K, Williams N. Assembly of the Trypanosoma brucei 60S ribosomal subunit nuclear export complex requires trypanosome-specific proteins P34 and P37. EUKARYOTIC CELL 2009; 8:77-87. [PMID: 18723605 PMCID: PMC2620753 DOI: 10.1128/ec.00234-08] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/15/2008] [Accepted: 08/14/2008] [Indexed: 12/30/2022]
Abstract
We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.
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Affiliation(s)
- Kimberly Prohaska
- Department of Microbiology and Immunology & Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, New York 14214, USA
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21
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Regmi S, Rothberg KG, Hubbard JG, Ruben L. The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis. Mol Microbiol 2008; 70:724-45. [PMID: 18786142 PMCID: PMC2581647 DOI: 10.1111/j.1365-2958.2008.06443.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow.
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Affiliation(s)
- Sandesh Regmi
- Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA
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22
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Hellman K, Prohaska K, Williams N. Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway. EUKARYOTIC CELL 2007; 6:2206-13. [PMID: 17921352 PMCID: PMC2168238 DOI: 10.1128/ec.00176-07] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/16/2007] [Accepted: 09/21/2007] [Indexed: 11/20/2022]
Abstract
We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.
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Affiliation(s)
- Kristina Hellman
- Department of Microbiology and Immunology and The Witebsky Center for Microbial Pathogenesis and Immunology, 253 Biomedical Research Building, University at Buffalo, Buffalo, New York 14214, USA
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23
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Elucidating the role of C/D snoRNA in rRNA processing and modification in Trypanosoma brucei. EUKARYOTIC CELL 2007; 7:86-101. [PMID: 17981991 DOI: 10.1128/ec.00215-07] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2'-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNA-processing and modification defects that caused lethality. Systematic mapping of 2'-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.
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24
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Hellman KM, Ciganda M, Brown SV, Li J, Ruyechan W, Williams N. Two trypanosome-specific proteins are essential factors for 5S rRNA abundance and ribosomal assembly in Trypanosoma brucei. EUKARYOTIC CELL 2007; 6:1766-72. [PMID: 17715362 PMCID: PMC2043393 DOI: 10.1128/ec.00119-07] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/11/2007] [Accepted: 08/14/2007] [Indexed: 11/20/2022]
Abstract
We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.
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Affiliation(s)
- Kristina M Hellman
- Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214, USA
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25
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Saveria T, Halbach A, Erdmann R, Volkmer-Engert R, Landgraf C, Rottensteiner H, Parsons M. Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes. EUKARYOTIC CELL 2007; 6:1439-49. [PMID: 17586720 PMCID: PMC1951143 DOI: 10.1128/ec.00084-07] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.
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Affiliation(s)
- Tracy Saveria
- Seattle Biomedical Research Institute, Seattle, WA 98109, USA
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26
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Boucher N, Dacheux D, Giroud C, Baltz T. An essential cell cycle-regulated nucleolar protein relocates to the mitotic spindle where it is involved in mitotic progression in Trypanosoma brucei. J Biol Chem 2007; 282:13780-90. [PMID: 17322293 DOI: 10.1074/jbc.m700780200] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G(1) cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G(2)/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N(*)2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.
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Affiliation(s)
- Nathalie Boucher
- Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR/CNRS-5234, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France.
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27
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Saveria T, Kessler P, Jensen BC, Parsons M. Characterization of glycosomal RING finger proteins of trypanosomatids. Exp Parasitol 2006; 116:14-24. [PMID: 17188680 PMCID: PMC1976121 DOI: 10.1016/j.exppara.2006.11.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2006] [Revised: 10/10/2006] [Accepted: 11/07/2006] [Indexed: 11/30/2022]
Abstract
The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.
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Affiliation(s)
- Tracy Saveria
- Seattle Biomedical Research Institute, 307 Westlake Avenue N., Seattle, WA 98109, USA
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Jensen BC, Kifer CT, Brekken DL, Randall AC, Wang Q, Drees BL, Parsons M. Characterization of protein kinase CK2 from Trypanosoma brucei. Mol Biochem Parasitol 2006; 151:28-40. [PMID: 17097160 PMCID: PMC1790856 DOI: 10.1016/j.molbiopara.2006.10.002] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2006] [Revised: 09/30/2006] [Accepted: 10/02/2006] [Indexed: 11/18/2022]
Abstract
CK2 is a ubiquitous but enigmatic kinase. The difficulty in assigning a role to CK2 centers on the fact that, to date, no biologically relevant modulator of its function has been identified. One common theme revolves around a constellation of known substrates involved in growth control, compatible with its concentration in the nucleus and nucleolus. We had previously described the identification of two catalytic subunits of CK2 in Trypanosoma brucei and characterized one of them. Here we report the characterization of the second catalytic subunit, CK2alpha', and the identification and characterization of the regulatory subunit CK2beta. All three subunits are primarily localized to the nucleolus in T. brucei. We also show that CK2beta interacts with the nucleolar protein NOG1, adding to the interaction map which previously linked CK2alpha to the nucleolar protein NOPP44/46, which in turn associates with the rRNA binding protein p37. CK2 activity has four distinctive features: near equal affinity for GTP and ATP, heparin sensitivity, and stimulation by polyamines and polybasic peptides. Sequence comparison shows that the parasite orthologues have mutations in residues previously mapped as important in specifying affinity for GTP and stimulation by both polyamines and polybasic peptides. Studies of the enzymatic activity of the T. brucei CK2s show that both the affinity for GTP and stimulation by polyamines have been lost and only the features of heparin inhibition and stimulation by polybasic peptides are conserved.
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Affiliation(s)
- Bryan C Jensen
- Seattle Biomedical Research Institute, 307 Westlake Ave N,
Suite 500, Seattle, WA 98108-5219 USA
| | - Charles T Kifer
- Seattle Biomedical Research Institute, 307 Westlake Ave N,
Suite 500, Seattle, WA 98108-5219 USA
| | - Deirdre L Brekken
- Seattle Biomedical Research Institute, 307 Westlake Ave N,
Suite 500, Seattle, WA 98108-5219 USA
| | - Amber C Randall
- Seattle Biomedical Research Institute, 307 Westlake Ave N,
Suite 500, Seattle, WA 98108-5219 USA
- Department of Pathobiology and
| | - Qin Wang
- Seattle Biomedical Research Institute, 307 Westlake Ave N,
Suite 500, Seattle, WA 98108-5219 USA
| | - Becky L. Drees
- Department of Genetics and Howard Hughes Medical Institute,
University of Washington, Seattle, WA 98195 USA
| | - Marilyn Parsons
- Seattle Biomedical Research Institute, 307 Westlake Ave N,
Suite 500, Seattle, WA 98108-5219 USA
- Department of Pathobiology and
- *Corresponding author.
, tell: +1-206-256-7315,
FAX: +1-206-256-7229
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Kelly S, Singleton W, Wickstead B, Ersfeld K, Gull K. Characterization and differential nuclear localization of Nopp140 and a novel Nopp140-like protein in trypanosomes. EUKARYOTIC CELL 2006; 5:876-9. [PMID: 16682465 PMCID: PMC1459678 DOI: 10.1128/ec.5.5.876-879.2006] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/20/2005] [Accepted: 03/08/2006] [Indexed: 11/20/2022]
Abstract
Trypanosomatids possess two homologues of Nopp140: a canonical Nopp140 and a Nopp140-like protein (TbNoLP) in which a GAR domain replaces the C-terminal SRP40 domain. Both are phosphorylated and coimmunoprecipitate with RNA polymerase I. Each paralogue has a distinct subnuclear localization, and depletion of TbNoLP produces an enlarged nucleolus in which TbNopp140-containing regions disperse. The restricted occurrence pattern of NoLP proteins reflects an intriguing convergence in evolution, suggestive of a function in nucleoplasmic small nucleolar ribonucleoprotein shuttling.
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Affiliation(s)
- S Kelly
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom
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Decuypere S, Vandesompele J, Yardley V, De Donckeri S, Laurent T, Rijal S, Llanos-Cuentas A, Chappuis F, Arevalo J, Dujardin JC. Differential polyadenylation of ribosomal RNA during post-transcriptional processing in Leishmania. Parasitology 2005; 131:321-9. [PMID: 16178353 DOI: 10.1017/s0031182005007808] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The protozoan parasite Leishmania belongs to the most ancient eukaryotic lineages and this is reflected in several distinctive biological features, such as eukaryotic polycistronic transcription and RNA trans-splicing. The disclosure of this organism's unusual characteristics leads to a better understanding of the origin and nature of fundamental biological processes in eukaryotes. Here we report another unusual phenomenon as we demonstrate that precursor ribosomal RNA can be extensively polyadenylated during post-transcriptional processingt. Furthermore, we demonstrate that the degree of precursor rRNA polyadenylation is variable in different strains and in the different life-stages of a strain.
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Affiliation(s)
- S Decuypere
- Department of Parasitology, Unit of Molecular Parasitology, Prince Leopold Institute of Tropical Medicine, Antwerp B-2000, Belgium
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