Potential and application of abortive transcripts as a novel molecular marker of cancers

Abstract

Abortive transcript (AT) is a 2-19 nt long non-coding RNA that is produced in the abortive initiation stage. Abortive initiation was found to be closely related to RNA polymerase through in vitro experiments. Therefore, the distribution of AT length and the scale of abortive initiation are correlated to the promoter, discriminator, and transcription initiation sequence, and can be affected by transcription elongation factors. AT plays an important role in the occurrence and development of various diseases. Here we summarize the discovery of AT, the factors responsible for AT formation, the detection methods and biological functions of AT, to provide new clues for finding potential targets in the early diagnosis and treatment of cancers.

Key Words: Abortive transcript, Abortive initiation, RNA polymerase, Transcription, Tumor marker

Core Tip: Abortive transcript (AT), as a special non-coding RNA, is the transcription product of abortive initiation. Abortive initiation occurs before normal transcription initiation and may be influenced by many factors. If its expression can be monitored normally, it will be of great significance for diagnosis and treatment of cancer. Though there are many difficulties and challenges in the study of AT in diseases, in-depth exploration of the role and mechanism of AT in cancers will provide a new potential target for early diagnosis and treatment of cancers and clinical prognosis.

INTRODUCTION

Abortive transcript (AT), is a special non-coding RNA, which is the transcription product of abortive initiation. Abortive initiation is an essential step during transcription initiation, where short nascent RNAs are synthesized and released by RNA polymerase (RNAP)[1]. AT is closely related to the structure of RNAP and the initiation process of RNA transcription[2]. AT was identified in 1976, and has been extensively studied since then by domestic and international scholars. AT not only plays a role in transcription, but also can be used as a new marker for early diagnosis and treatment of tumors. Here we summarize the research progress of AT in recent years.

DISCOVERY OF ABORTIVE TRANSCRIPT

Abortive initiation generally occurs in the body. At the initiation stage of transcription, RNAP cannot escape from the promoter region, but repeatedly synthesizes RNA with a fragment size of < 10 nt. RNAP plays a real transcription role until the > 10 nt RNA is synthesized. ATs are 2-8 nt long, and were first identified in vitro in 1976. When the extension reaction of RNAP lacks both NTPs (CTP and UTP), the transcription system is blocked and nascent RNA is released[3]. However, when all four NTPs (ATP, GTP, CTP and UTP) are present, RNAP transcription produces RNAs of varying lengths, which was initially called “abortive RNA”. In 1980, Carpousis et al[4] officially confirmed the existence of ATs when they detected 2-6 nt long abortive RNA through in vitro experiments and named it “abortive transcript”. In addition, it was found that 2 nt AT accounted for 50% of all abortive products. In 2009, Goldman first detected AT in Escherichia coli (E. coli). His team used plasmid transformation to express the N25anti gene (a mutant of the T5 phage N25 gene that produces 11-19 nt AT) and detected 11-19 nt AT in E. coli for the first time using a lock-in probe.

Researchers have found that the occurrence of abortive initiation is related to RNAP through in vitro experiments[2,5]. The length distribution of AT and the scale of abortive initiation are related to the promoter, discriminator and transcription initiation sequence, and are influenced by transcription elongation factors[6,7]. For example, the chain loops between the functional domains of RNAP σ3 and σ4 are close to the active site of RNAP, and located in the expulsion channel of RNA products in the whole enzyme, which can prevent the extension of RNA products, thus leading to abortive initiation[8]. Studies have found that the length of AT produced by abortive initiation is typically 2-10 nt, while a few ATs can reach 19 nt[1,2]. These studies provided important evidence in an emerging field of research.

FACTORS INFLUENCING ABORTIVE TRANSCRIPT FORMATION

RNA polymerase

RNAP is a complex enzyme composed of multiple subunits, which catalyzes transcription and can perform various functions. The holoenzyme form of RNAP is α₂ββ’σ, which consists of five subunits. The α subunit is related to the formation of α₂ββ’, the tetramer core of RNAP, and can determine which genes are transcribed. The β subunit contains the binding site of nucleoside triphosphate, which can catalyze polymerization. The β’ subunit contains a binding site for the DNA template. The σ subunit can recognize the start position of transcription and facilitate stable binding of RNAP to the promoter site[9]. Enzymes without σ subunit are called core enzymes, which can only catalyze chain elongation but have no effect on initiation[10]. Different σ subunits can respond to different signals and environmental conditions to identify specific promoter sequences of different genomes[11]. In addition, the CRE pocket formed by the β subunit of RNAP can make contact with the +2G position of the non-template DNA strand. However, this contact has no direct impact on the escape of the RNAP promoter, so the synthesis efficiency of abortive products will not change significantly[12].

In addition, the distance between the leading edge of the RNAP and its downstream DNA can affect the synthesis of ATs, which leads to a small decrease in abortive initiation that is reproducible and can affect the length of abortive products[13].

Promoter and promoter escape

Promoter is a region of the DNA sequence that can activate RNAP. It is located upstream of the 5’ end of a structural gene, which enables accurate binding of the RNAP to the template DNA and has specificity of transcription initiation[9]. Promoters T7A1, T7A2, T7A3 and T7D exist on isolated restriction fragments of the phage template[14], and all three phage promoters T7A1, T5N25 and T5N25 (antiDSR) can produce numerous ATs in the late stage of transcription initiation. DNA signals in the promoter recognition region and the initial transcription sequence region are key factors affecting the production of these ATs[15]. In addition, the initial T7N25 transcription sequence can undergo certain mutations that prolong abortive initiation and release 16-19 nt abortive transcripts. Among them, the hexamers at -35 and -10 positions can be altered, and the 17 base pairs in the middle gradually move away from the mutation, which inhibits the production of 16-19 nt long ATs[16]. In addition, when the promoter, RNAP, and 7-9 nt RNA product form a complex, abortive initiation can be inhibited to a certain extent and the synthesis rate of long RNA products can be increased[17]. Thus, the initiation of transcription is a critical stage of gene expression, which involves the interaction of RNAP with the promoter. The structure of the promoter affects its affinity for RNAP, and thus the level of gene expression.

In late transcription, promoter escape is the main biochemical reaction. In the abortive initiation curled model, RNAP is fixed on the promoter at the initiation stage of transcription and immobile. Instead, DNA downstream of the polymerase is collected into the polymerase, and the extension stage of transcription begins after the RNAP escapes from the promoter[18]. The strong interaction between RNAP and promoter recognition region can reduce the rate of promoter escape, thereby promoting abortive initiation[19]. Studies have shown that lacUV5 is the first escape rate-limiting promoter. By measuring the ratio of synthesized invalid RNA to productive RNA, it was found that lacUV5 promoter participates in more invalid synthesis and is more difficult to escape than T7A1 promoter[17].

Transcription factor Gre

RNAP can be involved in abortive initiation in vitro and synthesizes 2-15 nt transcripts. Hybridization with lock-in nucleic acid probes has shown that AT production can be directly detected in vivo. Gre transcription elongation factors (GreA and GreB) can combine with pore structures of secondary channels to enhance RNA cleavage by polymerases[20], which can reduce the occurrence of abortive initiation and affect the length of AT[21-23]. Further studies demonstrated that abortive initiation in vivo could reflect promoter strength and RNAP function and was regulated by GreA. E.coli GreA can also induce cleavage of transcripts as short as 4 nt at the initiation stage, thereby inhibiting the generation of ATs[24]. In addition, reducing the number of ATs at 6-10 nt and 11-15 nt and supplementing the transcription cycle with GreB protein, while the number of ATs at 2-5 nt and 16-20 nt was unchanged, increased RNA product synthesis by 2-5 times. Among them, 16-20 nt long ATs were not affected by GreB, suggesting that they were not products of RNAP backdating[23]. In addition, Gre may also affect promoter escape. Some studies have investigated the factors influencing promoter escape of E. coli RNAP, involving phage promoters T7A1, T5N25 and T5N25 (antiDSR). In vitro, the addition of E. coli transcription cleavage factor GreA or GreB can significantly increase the clearance rate of T5N25 (antiDSR), but has little effect on normal T7A1 and T5N25 regulated transcription. In vivo experiments with E. coli GreA and GreB knockout strains also showed that Gre factor derived from T5N25 (antiDSR) had a stimulating effect on transcription[7].

DETECTION METHODS FOR ABORTIVE TRANSCRIPTS

Detection of the special structure, function and biological role of AT depends on the advancements in biotechnology and new methods. At present, there are four main methods to detect short ATs.

Autoradiography technique for AT detection

It is an in vitro transcription technique based on a specific transcription template and p32-labeled NTP. This method can only detect the length and content of AT by autoradiography in vitro. However, it cannot identify the sequence of AT, or conduct qualitative and quantitative analyses of naturally produced ATs in the body[1].

The probe method

Since AT is a type of short-stranded RNA, it can be prolonged or the stability of its binding to the probe can be enhanced when using a locked nucleic acid probe. For example, SYBR Green is economical[25], rapid and highly sensitive, and TaqMan-MGB is highly specific, accurate and reliable[26]. Goldman et al[2] performed direct detection of 11 nt AT produced by T5 phage N25 gene with promoter mutation with a nucleic acid probe, which proved for the first time that plasmids could produce ATs in the body. However, due to technical difficulties and the special structure of AT, the above methods cannot achieve specific qualitative and quantitative analyses. Meanwhile, the existence of naturally generated ATs < 10 nt in the body has not been confirmed experimentally.

BSH-ABC model

According to the base packing hybridization principle, a base packing hybridization assisted ligation reaction is designed, that is, a ssDNA probe A and two auxiliary RNA strands B and C are designed to provide strong base packing force as well as prolong ATs. This method is called BSH-ABC mode[27]. After the extension of ATs in BSH-ABC mode, real-time reverse transcriptase-polymerase chain reaction and TaqMan-MGB probe can be used for the specific detection of ATs of different lengths with high sensitivity. However, since ATs produced in the body are not modified by fluorophores, BSH-ABC mode also has disadvantages in detecting ATs.

BSH-ABCD model

Based on the principle and technology of base packing hybridization and strand displacement reaction, studies have further explored the simultaneous and stable combination of two 8 nt oligonucleotide chains using their own double helix structure, which is called BSH-ABCD mode[28]. This method permits quantitative detection of ATs to some extent, but needs to be further explored.

BIOLOGICAL FUNCTIONS OF ABORTIVE TRANSCRIPTS

AT inhibits transcription

ATs could not only inhibit the transcription of the parent gene, but also interfere with the transcription of other related genes, and the inhibition rate was up to 7.5 times. The interference ability of ATs with different lengths and different types of genes is different. The length distribution of ATs and the scale of abortive initiation are related to the promoter, discriminator and transcription initiation sequence, and influenced by transcription elongation factors. ATs may inhibit transcription by interacting with RNAP[29]. Abortive initiation can affect the transcription termination of phage T7 gene 10. ATs derived from promoter φ10 can exhibit a trans-acting anti-termination activity against terminator Tφ. When the abortive initiation cycle of T7 RNAP on φ10 begins, oligomeric and polymeric short RNAs of G can be generated, which can specifically sequestrate the 5 nt and 6 nt C+U extension sequences and ultimately interfere with the formation of the terminator hairpin. This anti-termination activity depends on sequence-specific hybridization of ATs to the 5’-end of the half TφRNA. In vivo, excessive accumulation of RNAP and loss of ribonuclease can increase the AT content and enhance its anti-termination activity. In E. coli T7 infection, the anti-termination activity of ATs against Tφ promotes the expression of downstream promoter-free genes 11 and 12. Other studies have shown that abortive initiation can also act as a checkpoint for promoter proofreading and structural transformation, thereby regulating gene expression[30].

AT is involved in the synthesis of DNA and RNA

Transcription initiation in all cells is generally thought to occur as a function of NTP alone. However, it is well known that prokaryotic and eukaryotic RNAPs can initiate transcription in vitro using 2-8 nt long oligonucleotides. Goldman et al[31] found that using sequences in the 2-4 nt range are complementary to the coding strand. In addition, the artificially synthesized ATs with the 5’-end between -3 and +1, and the 3’-end between +1 and +3 of this segment can be used as primers to initiate transcription.

AT CAN BE USED AS A NOVEL MOLECULAR MARKER OF DISEASES

Liver injury

Yan et al[32] used high-throughput sequencing to detect the expression profile of 8 nt-long ATs in the serum of mice with or without acute liver injury induced by carbon tetrachloride. The results showed a total of 661 ATs in the experimental group and the control group, among which 16 ATs were differentially expressed in the two groups. Quantitative polymerase chain reaction was used to verify the high-throughput sequencing results, and the results of both techniques were consistent. The source of these 16 differentially expressed ATs was traced, and indicating that these 16 ATs may be derived from genes related to liver injury. Thus, AT may become a novel molecular marker of liver injury.

Ovarian cancer

CA125 protein has been identified as a biomarker for various cancers. The gene muc16 expressing CA125 protein can release numerous relevant ATs at the transcription initiation stage. Some studies have successfully detected the expression of muc16 related ATs[28]. Felder et al[33] found that CA125 was a repeat peptide epitope of muc16 in ovarian cancer, which can promote cancer cell proliferation and inhibit anticancer immune response. Muc16 is expressed in non-mucinous epithelial ovarian cancer[34]. It has become the most widely used and biomarker in the screening of ovarian cancer.

Pancreatic cancer

Haridas et al[35] detected the expression pattern of muc16 in pancreatic cancer tissues. The results showed that muc16 was not expressed in normal pancreas, but was significantly up-regulated in pancreatic cancer and pancreatitis tissues. These results suggest that muc16 may play a role in the progression and metastasis of pancreatic cancer.

Breast cancer

Lakshmanan et al[36] analyzed the expression of muc16 in breast cancer tissues and found that normal tissues did not express muc16, but 54% of breast cancer tissues showed positive muc16 expression. Their results indicate that muc16 is more commonly expressed in breast cancer tissues, and plays an important role in breast cancer occurrence and development.

Lung cancer

Muc16 is highly expressed in patients with multiple brain metastases from non-small cell lung cancer (NSCLC) and is associated with poor prognosis[37]. Other studies have shown that muc16 is elevated in serum samples from patients with stage I NSCLC, and may be a good biomarker for lung cancer[38].

Liver cancer

Xia et al[39] found that GPC3 is a differentially expressed gene in hepatocellular carcinoma that produces numerous ATs during the transcription process, and the stage and quantity of AT production can predict the efficiency and results of normal transcription to a certain extent. A recent study found that REXO2, a key enzyme responsible for cleaving the short fragment RNA of ATs[40,41], is closely related to the occurrence and development of liver cancer, and can promote the proliferation, migration and invasion of liver cancer cells[42].

Given these preliminary findings on the roles of ATs in various diseases, ATs have the potential to become biomarkers for cancer diagnosis and treatment. At present, there are several markers that can be used for cancer diagnosis. Some commonly used cancer markers are summarized in Tables 1, 2 and 3[43-65]. Therefore, exploring new, non-invasive, highly sensitive and specific early detection methods, and systematically and rigorously exploring the biological functions and mechanism of cancers, will provide new biomarkers for the early diagnosis and treatment of cancers.

Table 1 Protein tumor biomarkers.

Biomarker	Alteration	Related tumor	Detection method	Advantage	Disadvantage	Ref.
AFP	Up-regulation	Primary liver cancer, viral hepatitis, liver cirrhosis, gonad embryonic tumor, etc.	Blood	The most sensitive and specific index for early diagnosis of primary liver cancer, and suitable for large-scale census	For patients with early liver cancer, the detection rate of AFP is low and the misdiagnosis rate is high	[43]
CEA	Up-regulation	Colon cancer, rectal cancer, pancreatic cancer, gastric cancer, lung cancer, etc.	Blood	A broad-spectrum tumor marker	The specificity is not strong, the sensitivity is not high, and the early diagnosis of tumor is not obvious	[44-46]
PSA	Up-regulation	Prostate cancer	Blood	Avoid some unnecessary biopsies	Benign prostatic hyperplasia and prostatitis can also show positive PSA	[47]
SCCA	Up-regulation	Cervical cancer, lung squamous cell carcinoma, head and neck cancer, etc.	Blood and tissue	High specificity	Low sensitivity	[48-49]
CYFRA21-1	Up-regulation	Lung cancer, esophageal cancer, hepatocellular carcinoma, etc.	Blood	The sensitivity and specificity of the diagnosis for bladder cancer are good	Screening for lung cancer is not highly specific	[50-52]
TPA	Up-regulation	Lung cancer, breast cancer, ovarian cancer, bladder cancer	Blood	High detection rate for malignant tumors, and high sensitivity in the observation of curative effect	Has no correlation with tumor site or tissue type	[53-56]
HE4	Up-regulation	Ovarian cancer	Blood	High sensitivity and specificity for the diagnosis of ovarian cancer, especially in early ovarian cancer, and related to the stage and metastasis of ovarian cancer	A high positive rate in endometrial cancer	[57]

AFP: Alpha-fetoprotein; CEA: Carcinoembryonic antigen; PSA: Prostate specific antigen; SCCA: Squamous cell carcinoma antigen; CYFRA21-1: Cytokeratin 19 fragment; TPA: Tissue polypeptide antigen; HE4: Human epididymal protein 4.

Table 2 Glycochain antigen tumor biomarkers.

Biomarker	Alteration	Related tumor	Detection method	Advantage	Disadvantage	Ref.
CA125	Up-regulation	Ovarian cancer	Blood	High sensitivity of diagnosis	Poor specificity, nearly half of early cases are not elevated	[58]
CA15-3	Up-regulation	Lung cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, etc.	Blood	The most important specific marker for breast cancer	Low sensitivity in the early stages of breast cancer	[59]
CA19-9	Up-regulation	Pancreatic cancer, gallbladder cancer	Blood	The sensitivity and specificity can reach more than 90%	The elevation of CA19-9 is susceptible to many benign diseases	[60-61]
CA50	Up-regulation	Pancreatic cancer, colon cancer, liver cirrhosis, lung cancer	Blood	Double identify Lewis antigen negative and positive tumors, a broader broad-spectrum tumor marker than CA19-9	Inflammation of the gastrointestinal tract may cause mild or transient elevation of CA50	[62]

CA125: Glycochain antigen 125; CA15-3: Glycochain antigen 15-3; CA19-9: Glycochain antigen 19-9; CA50: Glycochain antigen 50.

Table 3 Enzymatic tumor biomarkers.

Biomarker	Alteration	Related tumor	Detection method	Advantage	Disadvantage	Ref.
NSE	Up-regulation	Small cell lung cancer	Blood	The diagnostic sensitivity can reach 80% and the specificity can reach 80%-90%	NSE alone is not sufficient to accurately differentiate SCLC from NSCLC	[63]
ProGRP	Up-regulation	Small cell lung cancer	Blood	High sensitivity and specificity	Insufficiency of kidney function can also lead to elevated ProGRP values	[64]
PAP	Up-regulation	Prostate cancer	Blood	An auxiliary index for tumor grading	Serum PAP may be elevated in some patients with benign prostatic hyperplasia	[65]

NSE: Neuron specific enolase; ProGRP: Gastrin releases peptide precursors; PAP: Prostatic acid phosphatase.

CONCLUSION

The normal transcription process may be affected by many factors and lead to abortive initiation, resulting in ATs. At is a short non-coding RNA that has been shown to affect the normal transcription process. Studies have demonstrated that ATs have the potential to become novel markers for liver injury and tumor, which can facilitate the early detection, diagnosis and treatment of cancer. For example, some lncRNAs, miRNAs and other non-coding RNAs have been confirmed as diagnostic and prognostic markers of cancers, and can affect their occurrence and development. As a special non-coding RNA, AT also has the potential to be a novel marker. At present, there are many challenges in the study of ATs in diseases. Given that REXO2 is a key enzyme required for AT cleavage and has a cancer-promoting effect, it is important to explore whether ATs play more biological roles and functions in the transcription process, and affect the occurrence, development and prognosis of cancers. Moreover, whether ATs can be employed for the early diagnosis of cancers needs further exploration, and its role and mechanism require rigorous study. Therefore, in-depth exploration of the role and mechanism of ATs in cancers will certainly provide a new strategy to search for potential targets for early diagnosis, treatment and clinical prognosis of cancers.