End Of RNA Research Articles

The highly conserved RNA-binding specificity of nucleocapsid protein facilitates the identification of drugs with broad anti-coronavirus activity

The binding of SARS-CoV-2 nucleocapsid (N) protein to both the 5′- and 3′-ends of genomic RNA has different implications arising from its binding to the central region during virion assembly. However, the mechanism underlying selective binding remains unknown. Herein, we performed the high-throughput RNA-SELEX (HTR-SELEX) to determine the RNA-binding specificity of the N proteins of various SARS-CoV-2 variants as well as other β-coronaviruses and showed that N proteins could bind two unrelated sequences, both of which were highly conserved across all variants and species. Interestingly, both sequences are virtually absent from the human transcriptome; however, they exhibit a highly enriched, mutually complementary distribution in the coronavirus genome, highlighting their varied functions in genome packaging. Our results provide mechanistic insights into viral genome packaging, thereby increasing the feasibility of developing drugs with broad-spectrum anti-coronavirus activity by targeting RNA binding by N proteins.

The role of circular RNA in Diabetic Nephropathy.

Diabetic nephropathy (DKD) is the most common chronic microvascular complication of diabetes. About 20%-40% of diabetics develop DKD, which eventually leads to chronic kidney failure. Although progress has been made in diagnosis and treatment tools, diabetic nephropathy is still a major clinical problem. In recent years, circular RNA (CircRNA) has become a research hotspot. CircRNA is a non-coding RNA formed by covalently closing the 5 'and 3' ends of the precursor RNA. CircRNA has powerful biological functions. CircRNA can regulate the expression of target genes through competitive binding with microRNA, thus playing the biological role of endogenous RNA (CeRNA). Many studies have shown that circRNAs plays an important role in malignant tumors, autoimmune system diseases, coronary heart disease and other diseases. More and more studies have shown that it can also be used as a biomarker of diabetes and diabetic nephropathy. This review summarizes the origin, classification, biogenesis and regulatory mechanisms of circRNAs. In addition, the pathogenesis and clinical significance of circRNAs as competing endogenous RNAs involved in diabetic nephropathy were also introduced. This will help us fully understand the pathological mechanism of diabetic nephropathy and develop new therapeutic targets or treatment options to improve the prognosis of patients with diabetic nephropathy.

Mechanism for the genomic and functional evolution of the MIR2118 family in the grass lineage.

The MIR2118 family has undergone tremendous expansion in the grass lineage, in which the miRNA targets numerous noncoding PHAS loci to produce 21-nt phased small interfering RNAs (phasiRNAs) involved in male fertility. However, the evolutionary trajectory of the grass MIR2118 genes and the functions of phasiRNAs have not yet been fully elucidated. We conducted comparative genomic, molecular evolution, expression and parallel analysis of RNA ends (PARE) analyses of MIR2118 and the miR2118-mediated regulatory pathway in grasses, focusing on Oryza sativa. In total, 617 MIR2118 and eight MIR1859 novel members were identified. Phylogenetic analyses showed that grass MIR2118 genes form a distinct clade from the MIR482/2118 genes of nongrass species. We reconstructed hypothetical evolutionary histories of the grass MIR2118 clusters and its MIR1859 variants, and examined the polycistronic composition and the differential expression of the osa-MIR2118 clusters. PARE data showed that osa-miR2118 might also direct the cleavage of some protein-coding gene transcripts. Importantly, we found that PARE analysis is inherently prone to false-positive target predictions when a large number of small RNAs, such as phasiRNAs, are analysed. Our results revealed the evolution and diversification of the MIR2118 family, and provide new insights into the functions of phasiRNAs in the grasses.

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type.

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3′ end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA–mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies.

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

Hepatitis Delta Virus Genome RNA Synthesis Initiates at Position 1646 with a Nontemplated Guanosine.

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense single-stranded RNA (ssRNA) virus that produces three RNAs in infected cells, genome, antigenome, and mRNA; the latter encodes hepatitis delta antigen (HDAg), the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5' end of the genome, including the synthesis start site and its chemical composition, is not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling-circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling the detection of the 5' end of the genome RNA. The 5' end of this RNA is capped, as expected for a polymerase II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how the synthesis of the viral RNA begins or even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed-hairpin-like structure of the viral RNA. The 5' end of the RNA is capped, as expected for polymerase II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses polymerase II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

The Cap-Binding Complex CBC and the Eukaryotic Translation Factor eIF4E: Co-Conspirators in Cap-Dependent RNA Maturation and Translation.

Simple SummaryTo produce the proteins needed for the cell to survive, the information in the DNA is converted to a mobile form known as messenger RNA, which exits the cell nucleus and binds to machines that convert RNAs into proteins in a process referred to as translation. Importantly, the RNA message can be altered at any point in its journey prior to translation. These alterations constitute changes to the chemical nature of the messenger RNA and modulate the ability of mRNAs to be converted into proteins and even lead to the production of proteins with different functionalities than those encoded by their original forms. Here we provide a conceptual framework for the integration of these mRNA maturation steps with translation with a focus on the proteins that escort these mRNAs through these steps and into the translation machines. We discuss the relevance to cancer and therapeutic strategies to target these in malignancy.The translation of RNA into protein is a dynamic process which is heavily regulated during normal cell physiology and can be dysregulated in human malignancies. Its dysregulation can impact selected groups of RNAs, modifying protein levels independently of transcription. Integral to their suitability for translation, RNAs undergo a series of maturation steps including the addition of the m7G cap on the 5′ end of RNAs, splicing, as well as cleavage and polyadenylation (CPA). Importantly, each of these steps can be coopted to modify the transcript signal. Factors that bind the m7G cap escort these RNAs through different steps of maturation and thus govern the physical nature of the final transcript product presented to the translation machinery. Here, we describe these steps and how the major m7G cap-binding factors in mammalian cells, the cap binding complex (CBC) and the eukaryotic translation initiation factor eIF4E, are positioned to chaperone transcripts through RNA maturation, nuclear export, and translation in a transcript-specific manner. To conceptualize a framework for the flow and integration of this genetic information, we discuss RNA maturation models and how these integrate with translation. Finally, we discuss how these processes can be coopted by cancer cells and means to target these in malignancy.

Systematic Characterization of MicroRNA Processing Modes in Plants With Parallel Amplification of RNA Ends.

In plants, the RNase III-type enzyme Dicer-like 1 (DCL1) processes most microRNAs (miRNAs) from their primary transcripts called pri-miRNAs. Four distinct processing modes (i.e., short base to loop, sequential base to loop, short loop to base, and sequential loop to base) have been characterized in Arabidopsis, mainly by the Specific Parallel Amplification of RNA Ends (SPARE) approach. However, SPARE is a targeted cloning method which requires optimization of cloning efficiency and specificity for each target. PARE (Parallel Amplification of RNA Ends) is an untargeted method per se and is widely used to identify miRNA mediated target slicing events. A major concern with PARE in characterizing miRNA processing modes is the potential contamination of mature miRNAs. Here, we provide a method to estimate miRNA contamination levels and showed that most publicly available PARE libraries have negligible miRNA contamination. Both the numbers and processing modes detected by PARE were similar to those identified by SPARE in Arabidopsis. PARE also determined the processing modes of 36 Arabidopsis miRNAs that were unexplored by SPARE, suggesting that it can complement the SPARE approach. Using publicly available PARE datasets, we identified the processing modes of 36, 91, 90, and 54 miRNAs in maize, rice, soybean, and tomato, respectively, and demonstrated that the processing mode was conserved overall within each miRNA family. Through its power of tracking miRNA processing remnants, PARE also facilitated miRNA characterization and annotation.

Reprogramming of RNA silencing triggered by cucumber mosaic virus infection in Arabidopsis

BackgroundRNA silencing has an important role mediating sequence-specific virus resistance in plants. The complex interaction of viruses with RNA silencing involves the loading of viral small interfering RNAs (vsiRNAs) into its host ARGONAUTE (AGO) proteins. As a side effect of their antiviral activity, vsiRNAs loading into AGO proteins can also mediate the silencing of endogenous genes. Here, we analyze at the genome-wide level both aspects of the interference of cucumber mosaic virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana.ResultsWe observe CMV-derived vsiRNAs affect the levels of endogenous sRNA classes. Furthermore, we analyze the incorporation of vsiRNAs into AGO proteins with a described antiviral role and the viral suppressor of RNA silencing (VSR) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. Interestingly, vsiRNAs represent a substantial percentage of AGO-loaded sRNAs and displace other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affect the expression of the genes they derive from. Additionally, we analyze how vsiRNAs incorporate into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing, which allow us to identify vsiRNA-targeted genes genome-wide.ConclusionsThis work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme.

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5′ triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

Salmonella Genomic Island 1 requires a self-encoded small RNA for mobilization.

The SGI1‐family elements that are specifically mobilized by the IncA‐ and IncC‐family plasmids are important vehicles of antibiotic resistance among enteric bacteria. Although SGI1 exploits many plasmid‐derived conjugation and regulatory functions, the basic mobilization module of the island is unrelated to that of IncC plasmids. This module contains the oriT and encodes the mobilization proteins MpsA and MpsB, which belong to the tyrosine recombinases and not to relaxases. Here we report an additional, essential transfer factor of SGI1. This is a small RNA deriving from the 3′‐end of a primary RNA that can also serve as mRNA of ORF S022. The functional domain of this sRNA named sgm‐sRNA is encoded between the mpsA gene and the oriT of SGI1. Terminator‐like sequence near the promoter of the primary transcript possibly has a regulatory function in controlling the amount of full‐length primary RNA, which is converted to the active sgm‐sRNA through consecutive maturation steps influenced by the 5′‐end of the primary RNA. The mobilization module of SGI1 seems unique due to its atypical relaxase and the newly identified sgm‐sRNA, which is required for the horizontal transfer of the island but appears to act differently from classical regulatory sRNAs.

Template requirements of Zika RNA polymerase during in vitro RNA synthesis from the 3′-end of virus minus-strand RNA

As ZIKV continues to spread, many “unknowns” remain and research is needed to advance the understanding of this important pathogen. Viral RNA dependent-RNA polymerases (RdRp) are validated targets for inhibitors of the replication of several viruses. Several studies have set up in vitro enzymatic assays of the RdRp of the Zika virus for testing of candidate inhibitors. While most of these studies use short synthetic polymers, we have shown in a previous work that the Zika polymerase domain is capable of a de novo synthesis of the viral genome using the natural viral RNA as template. Here we have studied the role of the sequences at the 3′end of the minus-strand RNA in the initiation of the RNA synthesis by the Zika isolated RdRp. Our results strongly suggest that the region containing the 105 first nucleotides from the 3′ end of the minus-strand RNA is important for initiation of the positive RNA synthesis. This indicates that this region displays all the primary and secondary structures to be efficiently recognized by the recombinant RdRp in vitro. Moreover, we show that the 46 nucleotides are sufficient to initiate RNA synthesis. In addition, the ZIKV polymerase domain poorly replicated the RNA of other RNA viruses and appeared highly selective for its own RNA.

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation.

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.

MiRNA-seq and clinical evaluation in multiple myeloma: miR-181a overexpression predicts short-term disease progression and poor post-treatment outcome.

Despite significant advances in multiple myeloma (MM) therapy, disease relapse and treatment resistance remain major obstacles in clinical management. Herein, we have studied the clinical utility of miRNAs in improving patients' risk-stratification and prognosis. miRNA-seq was performed in CD138+ plasma cells of MM, smoldering multiple myeloma (sMM) and monoclonal gammopathy of undetermined significance (MGUS) patients. The screening MM cohort consisted of 138 patients. miRNA levels of CD138+ plasma cells were quantified by RT-qPCR following 3'-end RNA polyadenylation. Disease progression and patients' death were used as clinical end-point events. Internal validation was conducted by bootstrap analysis. Clinical net benefit on disease prognosis was assessed by decision curve analysis. Kruykov et al. 2016 served as validation cohort (n = 151). miRNA-seq highlighted miR-181a to be upregulated in MM vs. sMM/MGUS, and R-ISS III vs. I patients. Screening and validation cohorts confirmed the significantly higher risk for short-term progression and worse survival of the patients overexpressing miR-181a. Multivariate models integrating miR-181a with disease established markers led to superior risk-stratification and clinical benefit for MM prognosis. CD138+ overexpression of miR-181a was strongly correlated with inferior disease outcome and contributed to superior prediction of MM patients early progression, supporting personalised prognosis and treatment decisions.

Distinct Evolutionary Profiles and Functions of microRNA156 and microRNA529 in Land Plants.

MicroRNA156 (miR156) and miR529 have high sequence similarity and recognize overlapping sites in the same target genes, SQUAMOSA promoter binding protein-like (SPL or SBP box) genes, making it difficult to accurately distinguish their roles in regulatory networks that affect numerous biological functions. Here, we collected data about miR156 and miR529 family members from representative land plants and performed sequence comparisons, phylogenetic analysis, small RNA sequencing, and parallel analysis of RNA ends (PARE) analysis to dissect their evolutionary and functional differences. Although miR156 and miR529 are highly similar, there are differences in their mismatch-sensitive regions, which are essential for target recognition. In land plants, miR156 precursors are conserved mainly within the hairpin region, whereas miR529 precursors are conserved outside the hairpin region, including both the 5’ and 3’ arms. Phylogenetic analysis showed that MIR156 and MIR529 evolved independently, through divergent evolutionary patterns. The two genes also exhibit different expression patterns, with MIR529 preferentially expressed in reproductive tissues and MIR156 in other tissues. PARE analysis revealed that miR156 and miR529 possess specific targets in addition to common targets in maize, pointing to functional differences between them. Based on our findings, we developed a method for the rapid identification of miR529 and miR156 family members and uncovered the evolutionary divergence of these families, providing insights into their different regulatory roles in plant growth and development.

β-CASP proteins removing RNA polymerase from DNA: when a torpedo is needed to shoot a sitting duck.

During the first step of gene expression, RNA polymerase (RNAP) engages DNA to transcribe RNA, forming highly stable complexes. These complexes need to be dissociated at the end of transcription units or when RNAP stalls during elongation and becomes an obstacle (‘sitting duck’) to further transcription or replication. In this review, we first outline the mechanisms involved in these processes. Then, we explore in detail the torpedo mechanism whereby a 5′–3′ RNA exonuclease (torpedo) latches itself onto the 5′ end of RNA protruding from RNAP, degrades it and upon contact with RNAP, induces dissociation of the complex. This mechanism, originally described in Eukaryotes and executed by Xrn-type 5′–3′ exonucleases, was recently found in Bacteria and Archaea, mediated by β-CASP family exonucleases. We discuss the mechanistic aspects of this process across the three kingdoms of life and conclude that 5′–3′ exoribonucleases (β-CASP and Xrn families) involved in the ancient torpedo mechanism have emerged at least twice during evolution.

Cap analysis of gene expression (CAGE) and noncoding regulatory elements.

Cap analysis of gene expression (CAGE) was developed to detect the 5' end of RNA. Trapping of the RNA 5'-cap structure enables the enrichment and selective sequencing of complete transcripts. Upscaled high-throughput versions of CAGE have enabled the genome-wide identification of transcription start sites, including transcriptionally active promoters and enhancers. CAGE sequencing can be exploited to draw comprehensive maps of active genomic regulatory elements in a cell type- and activation-specific manner. The cells of the immune system are among the best candidates to be analyzed in humans, since they are easily accessible. In this review, we discuss how CAGE data are instrumental for integrative analyses with quantitative trait loci and omics data, and their usefulness in the mechanistic interpretation of the effects of genetic variations over the entire human genome. Integrating CAGE data with the currently available omics information will contribute to better understanding of the genome-wide association study variants that lie outside of annotated genes, deepening our knowledge on human diseases, and enabling the targeted design of more specific therapeutic interventions.

TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization.

Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5′ ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5′ and 3′ ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5′ ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.

Two distinct pathways of RNA polymerase backtracking determine the requirement for the Trigger Loop during RNA hydrolysis.

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3′ end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3′ end of RNA within the active center. We show that the TL is required to transfer the 3′ end of misincorporated transcript from cleavage-inefficient ‘misincorporation site’ into the cleavage-efficient ‘backtracked site’, where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.

Feature selection for RNA cleavage efficiency at specific sites using the LASSO regression model in Arabidopsis thaliana

BackgroundRNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana.ResultsCleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model. The results indicated that whole RNA features were important for the CSsite value, in addition to features around cleavage sites. Whole RNA features related to the translation process and nucleotide frequency around cleavage sites were major determinants of cleavage efficiency. The results were verified in a model constructed using only sequence features, which showed that the prediction accuracy was similar to that determined using all features including the translation process, suggesting that cleavage efficiency can be predicted using only sequence information. The LASSO regression model was validated in exogenous genes, which showed that the model constructed using only sequence information can predict cleavage efficiency in both endogenous and exogenous genes.ConclusionsFeature selection using the LASSO regression model in A. thaliana identified 155 features. Correlation coefficients revealed that whole RNA features are important for determining cleavage efficiency in addition to features around the cleavage sites. The LASSO regression model can predict cleavage efficiency in endogenous and exogenous genes using only sequence information. The model revealed the significance of the effect of multiple determinants on cleavage efficiency, suggesting that sequence features are important for RNA degradation mechanisms in A. thaliana.