Role Of RNA Secondary Structure Research Articles

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

The Secondary Structure of Potato Spindle Tuber Viroid Determines Its Infectivity in Nicotiana benthamiana.

The function of RNAs is determined by their structure. However, studying the relationship between RNA structure and function often requires altering RNA sequences to modify the structures, which leads to the neglect of the importance of RNA sequences themselves. In our research, we utilized potato spindle tuber viroid (PSTVd), a circular-form non-coding infectious RNA, as a model with which to investigate the role of a specific rod-like structure in RNA function. By generating linear RNA transcripts with different start sites, we established 12 PSTVd forms with different secondary structures while maintaining the same sequence. The RNA secondary structures were predicted using the mfold tool and validated through native PAGE gel electrophoresis after in vitro RNA folding. Analysis using plant infection assays revealed that the formation of a correct rod-like structure is crucial for the successful infection of PSTVd. Interestingly, the inability of PSTVd forms with non-rod-like structures to infect plants could be partially compensated by increasing the amount of linear viroid RNA transcripts, suggesting the existence of additional RNA secondary structures, such as the correct rod-like structure, alongside the dominant structure in the RNA inoculum of these forms. Our study demonstrates the critical role of RNA secondary structures in determining the function of infectious RNAs.

Regulation of coronavirus nsp15 cleavage specificity by RNA structure.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had an enduring impact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (nsps) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established endonuclease function of nsp15, little is known about other determinants of its cleavage specificity. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS-CoV-1 3'UTR as a model for our structural studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus highly probable targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the GNRNA pentaloop of the RNA. Further investigation revealed that the position of uridine within the pentaloop also impacted nsp15 cleavage efficiency suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of RNA recognition by nsp15.

Aberrant splicing in human cancer: An RNA structural code point of view.

Alternative splicing represents an essential process that occurs widely in eukaryotes. In humans, most genes undergo alternative splicing to ensure transcriptome and proteome diversity reflecting their functional complexity. Over the last decade, aberrantly spliced transcripts due to mutations in cis- or trans-acting splicing regulators have been tightly associated with cancer development, largely drawing scientific attention. Although a plethora of single proteins, ribonucleoproteins, complexed RNAs, and short RNA sequences have emerged as nodal contributors to the splicing cascade, the role of RNA secondary structures in warranting splicing fidelity has been underestimated. Recent studies have leveraged the establishment of novel high-throughput methodologies and bioinformatic tools to shed light on an additional layer of splicing regulation in the context of RNA structural elements. This short review focuses on the most recent available data on splicing mechanism regulation on the basis of RNA secondary structure, emphasizing the importance of the complex RNA G-quadruplex structures (rG4s), and other specific RNA motifs identified as splicing silencers or enhancers. Moreover, it intends to provide knowledge on newly established techniques that allow the identification of RNA structural elements and highlight the potential to develop new RNA-oriented therapeutic strategies against cancer.

Deciphering the role of RNA structure in translation efficiency

BackgroundRNA secondary structure has broad impact on the fate of RNA metabolism. The reduced stability of secondary structures near the translation initiation site/start codon of the coding region promotes the efficiency of translation in both prokaryotic and eukaryotic species. However, the inaccuracy of in silico folding and the focus on the coding region limit our understanding of the global relationship between the whole mRNA structure and translation efficiency. Leveraging high-throughput RNA structure probing data in the transcriptome, we aim to systematically investigate the role of RNA structure in regulating translation efficiency.ResultsHere, we analyze the influences of hundreds of sequence and structural features on translation efficiency in the mouse embryonic stem cells (mESCs) and zebrafish developmental stages. Our findings reveal that overall in vivo RNA structure has a higher relative importance in predicting translation efficiency than in vitro RNA structure in both mESCs and zebrafish. Also, RNA structures in 3’ untranslated region (UTR) have much stronger influence on translation efficiency compared to those in coding regions or 5' UTR. Furthermore, strong alternation between in vitro and in vivo structures in 3' UTR are detected in highly translated mRNAs in mESCs but not zebrafish. Instead, moderate alteration between in vitro and in vivo RNA structures in the 5’ UTR and proximal coding regions are detected in highly translated mRNAs in zebrafish.ConclusionsOur results suggest the openness of the 3’ UTR promotes the translation efficiency in both mice and zebrafish, with the in vivo structure in 3’ UTR more important in mice than in zebrafish. This reveals a novel role of RNA secondary structure on translational regulation.

Expansion of the RNAStructuromeDB to include secondary structural data spanning the human protein-coding transcriptome

RNA plays vital functional roles in almost every component of biology, and these functional roles are often influenced by its folding into secondary and tertiary structures. An important role of RNA secondary structure is in maintaining proper gene regulation; therefore, making accurate predictions of the structures involved in these processes is important. In this study, we have expanded on our previous work that led to the creation of the RNAStructuromeDB. Unlike this previous study that analyzed the human genome at low resolution, we have now scanned the protein-coding human transcriptome at high (single nt) resolution. This provides more robust structure predictions for over 100,000 isoforms of known protein-coding genes. Notably, we also utilize the motif identification tool, ScanFold, to model structures with high propensity for ordered/evolved stability. All data have been uploaded to the RNAStructuromeDB, allowing for easy searching of transcripts, visualization of data tracks (via the IntegrativeGenomicsViewer or IGV), and download of ScanFold data—including unique highly-ordered motifs. Herein, we provide an example analysis of MAT2A to demonstrate the utility of ScanFold at finding known and novel secondary structures, highlighting regions of potential functionality, and guiding generation of functional hypotheses through use of the data.

The Role of RNA Secondary Structure in Regulation of Gene Expression in Bacteria.

Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation.

SMAtool reveals sequences and structural principles of protein-RNA interaction

Protein binding events on RNA are highly related to RNA secondary structure, which affects post-transcriptional regulation and translation. However, it remains challenging to describe the association between RNA secondary structure and protein binding events. Here, we present Structure Motif Analysis tool (SMAtool), a pipeline that integrates RNA secondary structure and protein binding site information to profile the binding structure preference of each protein. As an example of applying SMAtool, we extracted the RNA-structure and binding site information respectively from the DMS-seq and eCLIP-seq data of the K562 cell-line, and used SMAtool to analyze the structure motif of each RNA binding protein (RBP). This new approach provided results consistent with X-ray crystallography data from the protein data bank (PDB) database, demonstrating that it can help researchers investigate the structure preference of RBP, and understand the role of RNA secondary structure in gene expression. Availability and implementation: https://github.com/QuKunLab/SMAtool.

Role of RNA secondary structure in emergence of compartment specific hepatitis B virus immune escape variants.

AIMTo investigate the role of subgenotype specific RNA secondary structure in the compartment specific selection of hepatitis B virus (HBV) immune escape mutations.METHODSThis study was based on the analysis of the specific observation of HBV subgenotype A1 in the serum/plasma, while subgenotype A2 with G145R mutation in the peripheral blood leukocytes (PBLs). Genetic variability found among the two subgenotypes was used for prediction and comparison of the full length pregenomic RNA (pgRNA) secondary structure and base pairings. RNA secondary structures were predicted for 37 °C using the Vienna RNA fold server, using default parameters. Visualization and detailed analysis was done using RNA shapes program.RESULTSIn this analysis, using similar algorithm and conditions, entirely different pgRNA secondary structures for subgenotype A1 and subgenotype A2 were predicted, suggesting different base pairing patterns within the two subgenotypes of genotype A, specifically, in the HBV genetic region encoding the major hydrophilic loop. We observed that for subgenotype A1 specific pgRNA, nucleotide 358U base paired with 1738A and nucleotide 587G base paired with 607C. However in sharp contrast, in subgenotype A2 specific pgRNA, nucleotide 358U was opposite to nucleotide 588G, while 587G was opposite to 359U, hence precluding correct base pairing and thereby lesser stability of the stem structure. When the nucleotides at 358U and 587G were replaced with 358C and 587A respectively (as observed specifically in the PBL associated A2 sequences), these nucleotides base paired correctly with 588G and 359U, respectively.CONCLUSIONThe results of this study show that compartment specific mutations are associated with HBV subgenotype specific alterations in base pairing of the pgRNA, leading to compartment specific selection and preponderance of specific HBV subgenotype with unique mutational pattern.

Role of RNA Secondary Structure and Processing in Stability of the nifH1 Transcript in the Cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis ATCC 29413, aerobic nitrogen fixation occurs in micro-oxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of the large nif1 gene cluster, which is primarily under the control of the promoter for the first gene, nifB1. Strong expression of nifH1 requires the nifB1 promoter but is also controlled by RNA processing, which leads to increased nifH1 transcript stability. The processing of the primary nifH1 transcript occurs at the base of a predicted stem-loop structure that is conserved in many heterocystous cyanobacteria. Mutations that changed the predicted secondary structure or changed the sequence of the stem-loop had detrimental effects on the amount of nifH1 transcript, with mutations that altered or destabilized the structure having the strongest effect. Just upstream from the transcriptional processing site for nifH1 was the promoter for a small antisense RNA, sava4870.1. This RNA was more strongly expressed in cells grown in the presence of fixed nitrogen and was downregulated in cells 24 h after nitrogen step down. A mutant strain lacking the promoter for sava4870.1 showed delayed nitrogen fixation; however, that phenotype might have resulted from an effect of the mutation on the processing of the nifH1 transcript. The nifH1 transcript was the most abundant and most stable nif1 transcript, while nifD1 and nifK1, just downstream of nifH1, were present in much smaller amounts and were less stable. The nifD1 and nifK1 transcripts were also processed at sites just upstream of nifD1 and nifK1. In the filamentous cyanobacterium Anabaena variabilis, the nif1 cluster, encoding the primary Mo nitrogenase, functions under aerobic growth conditions in specialized cells called heterocysts that develop in response to starvation for fixed nitrogen. The large cluster comprising more than a dozen nif1 genes is transcribed primarily from the promoter for the first gene, nifB1; however, this does not explain the large amount of transcript for the structural genes nifH1, nifD1, and nifK1, which are also under the control of the distant nifB1 promoter. Here, we demonstrate the importance of a predicted stem-loop structure upstream of nifH1 that controls the abundance of nifH1 transcript through transcript processing and stabilization and show that nifD1 and nifK1 transcripts are also controlled by transcript processing.

Alternative splicing of TAF6: downstream transcriptome impacts and upstream RNA splice control elements.

The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ.

Finding the target sites of RNA‐binding proteins

RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201

The Role of RNA Secondary Structure in Viral Assembly

Some small icosahedral RNA viruses (e.g., MS2) require a specific packaging signal for the formation of virus particles. Others (e.g., cowpea chlorotic mottle virus (CCMV); Pariacoto virus (PaV)) are able to encapsidate a wide variety of RNAs, forming virus-like particles (VLPs) whose structure are only slightly different from that of the wild-type virus. We have determined the structure of the genomic RNA of satellite tobacco mosaic virus (STMV) in an in vitro transcript (see image below). This structure is very different from the structure of STMV RNA probed inside the virus by Schroeder et al. (BJ 101:167 (2011)), which consists of a string of stem-loops connected by single-stranded regions. We have also developed all-atom models for three viruses: PaV (using a hypothetical secondary structure and a non-viral sequence); STMV (using the true sequence and the Schroeder secondary structure); and MS2 (using the true sequence and a hypothetical secondary structure). In this talk, we discuss the implications of these secondary structures and three-dimensional models for the pathways of viral assembly.

Second-Site Suppression of RNase E Essentiality by Mutation of thedeaDRNA Helicase in Escherichia coli

Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.

MBNL1-RNA Interactions: Binding-Induced Rna Conformational Changes

The muscleblind-like protein 1 (MBNL1) is an RNA binding protein that regulates mRNA alternative splicing. It is implicated to be involved in myotonic dystrophy, an inherited multisystem disease, through interacting with poly(CUG) or poly(CCUG) RNA. In the disease state, MBNL1 is abnormally expressed and translocated, which leads to many mis-splicing events. MBNL1 can bind various RNA substrates, most of which share a stem-loop structure containing 5’YGCY3’(Y=U or C) motifs. Using FRET together with a combination of stopped-flow kinetic fluorescence measurements and gel shift assays, we have investigated the relationship between MBNL1-RNA interactions and RNA secondary structure. This work suggested that binding of MBNL1 destabilizes the RNA secondary structure and leads to a partial melting of the stem loop RNA. The RNA secondary structure stability is directly correlated with the kinetic and thermodynamic properties of the MBNL1-RNA complex. The data presented here provides insight into the role of RNA secondary structure in regulation of alternative splicing by MBNL1 and suggests new perspectives for drug development for myotonic dystrophy.

RNA structure and the mechanisms of alternative splicing

Alternative splicing is a widespread means of increasing protein diversity and regulating gene expression in eukaryotes. Much progress has been made in understanding the proteins involved in regulating alternative splicing, the sequences they bind to, and how these interactions lead to changes in splicing patterns. However, several recent studies have identified other players involved in regulating alternative splicing. A major theme emerging from these studies is that RNA secondary structures play an under appreciated role in the regulation of alternative splicing. This review provides an overview of the basic aspects of splicing regulation and highlights recent progress in understanding the role of RNA secondary structure in this process.

Characterization and prediction of mRNA polyadenylation sites in human genes

The accurate identification of potential poly(A) sites has contributed to all many studies with regard to alternative polyadenylation. The aim of this study was the development of a machine-learning methodology that will help to discriminate real polyadenylation signals from randomly occurring signals in genomic sequence. Since previous studies have revealed that RNA secondary structure in certain genes has significant impact, the authors tried to computationally pinpoint common structural patterns around the poly(A) sites and to investigate how RNA secondary structure may influence polyadenylation. This involved an initial study on the impact of RNA structure and it was found using motif search tools that hairpin structures might be important. Thus, it was propose that, in addition to the sequence pattern around poly(A) sites, there exists a widespread structural pattern that is also employed during human mRNA polyadenylation. In this study, the authors present a computational model that uses support vector machines to predict human poly(A) sites. The results show that this predictive model has a comparable performance to the current prediction tool. In addition, it was identified common structural patterns associated with polyadenylation using several motif finding programs and this provides new insight into the role of RNA secondary structure plays in polyadenylation.

GC content around splice sites affects splicing through pre-mRNA secondary structures

BackgroundAlternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens), mice (Mus musculus), fruit flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans) to further investigate this phenomenon.ResultsWe observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures.ConclusionAll of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

Role of RNA structure and protein factors in the control of HIV-1 splicing

Alternative splicing plays a key role in the production of numerous proteins by complex lentiviruses such as HIV-1. The study of HIV-1 RNA splicing has provided useful information not only about the physiology of the virus, but also about the general mechanisms that regulate mammalian pre-mRNA alternative splicing. Like all retroviruses, a fraction of HIV-1 transcripts remains intact to serve as genomic RNA and to code for Gag and Gag-Pol protein precursors. In addition, splicing is important for controlling the production of some viral proteins, which could otherwise have a negative effect on the infected cell. Here, we summarize how the utilization of HIV-1 splicing sites is limited by the binding of nuclear factors to cis-acting silencer elements, taking into account the role of RNA secondary structure in these mechanisms. We also describe how the poorly efficient HIV-1 acceptor sites are nevertheless activated by serine/arginine-rich proteins. Finally, we discuss how nuclear factors that interact with both the transcription and splicing machineries also participate in the control of HIV-1 RNA splicing.

Www.rnaworkbench.com: A new program for analyzing RNA interference

RNA interference (RNAi) has become an important tool to study and utilize gene silencing by introducing short interfering RNA (siRNA). In order to predict the most efficient siRNAs, a new software tool, RNA Workbench (RNAWB), has been designed and is freely available (after registration) on http://www.rnaworkbench.com. In addition to the standard selection rules, RNAWB includes the possibility of statistical analyses of the applied selection rules (criteria). The role of RNA secondary structures in the RNA interference process as well as the application of sequence rules are discussed to show the applicability of the software.