RNA Structurome Research Articles

DHX36 binding induces RNA structurome remodeling and regulates RNA abundance via m6A reader YTHDF1.

RNA structure constitutes a new layer of gene regulatory mechanisms. RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) is known to bind and unwind RNA G-quadruplex (rG4) structure but the transcriptome-wide RNA structure remodeling induced by DHX36 binding and the impact on RNA fate remain poorly understood. Here, we investigate the RNA structurome alteration induced by DHX36 depletion. Our findings reveal that DHX36 binding induces structural remodeling not only at the localized binding sites but also on the entire mRNA transcript most pronounced in 3'UTR regions. DHX36 binding increases structural accessibility at 3'UTRs which is correlated with decreased post-transcriptional mRNA abundance. Further analyses and experiments uncover that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding; and DHX36 induced structural changes may facilitate YTHDF1 binding to m6A sites leading to RNA degradation. Altogether, our findings uncover the structural remodeling effect of DHX36 binding and its impact on RNA abundance through regulating m6A dependent YTHDF1 binding.

The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic transcripts whose native structures remain unexplored, posing a gap in understanding mitochondrial gene expression. In this work, we devised the mitochondrial dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method and applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel the native mitochondrial messenger RNA (mt-mRNA) structurome in wild-type (WT) and leucine-rich pentatricopeptide repeat-containing protein (LRPPRC)-deficient cells. Our findings elucidate LRPPRC's role as a holdase contributing to maintaining mt-mRNA folding and efficient translation. mt-mRNA structural insights in WT mitochondria, coupled with metabolic labeling, unveil potential mRNA-programmed translational pausing and a distinct programmed ribosomal frameshifting mechanism. Our data define a critical layer of mitochondrial gene expression regulation. These mt-mRNA folding maps provide a reference for studying mt-mRNA structures in diverse physiological and pathological contexts.

Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seq.

RNA decay serves as a crucial mechanism for maintaining cellular homeostasis and regulating gene expression. Large-scale analyses indicate that altered rates of decay contribute significantly to changes in mRNA levels, with up to half of these changes attributed to decay. The regulation of RNA decay is, at least in part, through structured RNA elements, especially in the non-coding regions of the mRNAs. The development of next-generation sequencing, and in vivo chemical probing techniques has allowed for unprecedented understanding of RNA folding in vivo and genome-wide. To explore the RNA structure elements that are responsible for RNA cleavage, we need to capture the RNA structure before cleavage. In this method, we introduce a new experimental procedure called CAP-STRUCTURE-seq, a modified STRUCTURE-Seq approach combining with the enrichment of in intact mRNAs by the use of terminator exonuclease treatment (5'-Phosphate-Dependent Exonuclease) that digests RNA containing 5-monophosphate ends. This approach is designed to investigate the RNA structure for these intact RNAs, providing a means to study the impact of RNA structure on RNA decay in greater detail. This method can provide insights into the function of RNA structure in RNA decay and help advance our understanding of biological processes.

Probing the dynamic RNA structurome and its functions.

RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome - the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.

The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/U-rich transcriptome.

Plasmodium falciparum, a protozoan parasite and causative agent of human malaria, has one of the most A/T-biased genomes sequenced to date. This may give the genome and the transcriptome unusual structural features. Recent progress in sequencing techniques has made it possible to study the secondary structures of RNA molecules at the transcriptomic level. Thus, in this study we produced the in vivo RNA structurome of a protozoan parasite with a highly A/U-biased transcriptome. We showed that it is possible to probe the secondary structures of P. falciparum RNA molecules in vivo using two different chemical probes, and obtained structures for more than half of all transcripts in the transcriptome. These showed greater stability (lower free energy) than the same structures modelled in silico, and structural features appeared to influence translation efficiency and RNA decay. Finally, we compared the P. falciparum RNA structurome with the predicted RNA structurome of an A/U-balanced species, P. knowlesi, finding a bias towards lower overall transcript stability and more hairpins and multi-stem loops in P. falciparum. This unusual protozoan RNA structurome will provide a basis for similar studies in other protozoans and also in other unusual genomes.

Differential analysis of RNA structure probing experiments at nucleotide resolution: uncovering regulatory functions of RNA structure

RNAs perform their function by forming specific structures, which can change across cellular conditions. Structure probing experiments combined with next generation sequencing technology have enabled transcriptome-wide analysis of RNA secondary structure in various cellular conditions. Differential analysis of structure probing data in different conditions can reveal the RNA structurally variable regions (SVRs), which is important for understanding RNA functions. Here, we propose DiffScan, a computational framework for normalization and differential analysis of structure probing data in high resolution. DiffScan preprocesses structure probing datasets to remove systematic bias, and then scans the transcripts to identify SVRs and adaptively determines their lengths and locations. The proposed approach is compatible with most structure probing platforms (e.g., icSHAPE, DMS-seq). When evaluated with simulated and benchmark datasets, DiffScan identifies structurally variable regions at nucleotide resolution, with substantial improvement in accuracy compared with existing SVR detection methods. Moreover, the improvement is robust when tested in multiple structure probing platforms. Application of DiffScan in a dataset of multi-subcellular RNA structurome and a subsequent motif enrichment analysis suggest potential links of RNA structural variation and mRNA abundance, possibly mediated by RNA binding proteins such as the serine/arginine rich splicing factors. This work provides an effective tool for differential analysis of RNA secondary structure, reinforcing the power of structure probing experiments in deciphering the dynamic RNA structurome.

Recent advances in RNA structurome.

RNA structures are essential to support RNA functions and regulation in various biological processes. Recently, a range of novel technologies have been developed to decode genome-wide RNA structures and novel modes of functionality across a wide range of species. In this review, we summarize key strategies for probing the RNA structurome and discuss the pros and cons of representative technologies. In particular, these new technologies have been applied to dissect the structural landscape of the SARS-CoV-2 RNA genome. We also summarize the functionalities of RNA structures discovered in different regulatory layers—including RNA processing, transport, localization, and mRNA translation—across viruses, bacteria, animals, and plants. We review many versatile RNA structural elements in the context of different physiological and pathological processes (e.g., cell differentiation, stress response, and viral replication). Finally, we discuss future prospects for RNA structural studies to map the RNA structurome at higher resolution and at the single-molecule and single-cell level, and to decipher novel modes of RNA structures and functions for innovative applications.

Wheat in vivo RNA structure landscape reveals a prevalent role of RNA structure in modulating translational subgenome expression asymmetry

BackgroundPolyploidy, especially allopolyploidy, which entails merging divergent genomes via hybridization and whole-genome duplication (WGD), is a major route to speciation in plants. The duplication among the parental genomes (subgenomes) often leads to one subgenome becoming dominant over the other(s), resulting in subgenome asymmetry in gene content and expression. Polyploid wheats are allopolyploids with most genes present in two (tetraploid) or three (hexaploid) functional copies, which commonly show subgenome expression asymmetry. It is unknown whether a similar subgenome asymmetry exists during translation. We aim to address this key biological question and explore the major contributing factors to subgenome translation asymmetry.ResultsHere, we obtain the first tetraploid wheat translatome and reveal that subgenome expression asymmetry exists at the translational level. We further perform in vivo RNA structure profiling to obtain the wheat RNA structure landscape and find that mRNA structure has a strong impact on translation, independent of GC content. We discover a previously uncharacterized contribution of RNA structure in subgenome translation asymmetry. We identify 3564 single-nucleotide variations (SNVs) across the transcriptomes between the two tetraploid wheat subgenomes, which induce large RNA structure disparities. These SNVs are highly conserved within durum wheat cultivars but are divergent in both domesticated and wild emmer wheat.ConclusionsWe successfully determine both the translatome and in vivo RNA structurome in tetraploid wheat. We reveal that RNA structure serves as an important modulator of translational subgenome expression asymmetry in polyploids. Our work provides a new perspective for molecular breeding of major polyploid crops.

Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (−) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.

Genome-scale deconvolution of RNA structure ensembles.

RNA structure heterogeneity is a major challenge when querying RNA structures with chemical probing. We introduce DRACO, an algorithm for the deconvolution of coexisting RNA conformations from mutational profiling experiments. Analysis of the SARS-CoV-2 genome using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and DRACO, identifies multiple regions that fold into two mutually exclusive conformations, including a conserved structural switch in the 3' untranslated region. This work may open the way to dissecting the heterogeneity of the RNA structurome.

Specificity of mRNA Folding and Its Association with Evolutionarily Adaptive mRNA Secondary Structures

The secondary structure is a fundamental feature of both non-coding RNAs (ncRNAs) and messenger RNAs (mRNAs). However, our understanding of the secondary structures of mRNAs, especially those of the coding regions, remains elusive, likely due to translation and the lack of RNA-binding proteins that sustain the consensus structure like those binding to ncRNAs. Indeed, mRNAs have recently been found to adopt diverse alternative structures, but the overall functional significance remains untested. We hereby approach this problem by estimating the folding specificity, i.e., the probability that a fragment of an mRNA folds back to the same partner once refolded. We show that the folding specificity of mRNAs is lower than that of ncRNAs and exhibits moderate evolutionary conservation. Notably, we find that specific rather than alternative folding is likely evolutionarily adaptive since specific folding is frequently associated with functionally important genes or sites within a gene. Additional analysis in combination with ribosome density suggests the ability to modulate ribosome movement as one potential functional advantage provided by specific folding. Our findings reveal a novel facet of the RNA structurome with important functional and evolutionary implications and indicate a potential method for distinguishing the mRNA secondary structures maintained by natural selection from molecular noise.

ChiRA: an integrated framework for chimeric read analysis from RNA-RNA interactome and RNA structurome data.

BackgroundWith the advances in next-generation sequencing technologies, it is possible to determine RNA-RNA interaction and RNA structure predictions on a genome-wide level. The reads from these experiments usually are chimeric, with each arm generated from one of the interaction partners. Owing to short read lengths, often these sequenced arms ambiguously map to multiple locations. Thus, inferring the origin of these can be quite complicated. Here we present ChiRA, a generic framework for sensitive annotation of these chimeric reads, which in turn can be used to predict the sequenced hybrids.ResultsGrouping reference loci on the basis of aligned common reads and quantification improved the handling of the multi-mapped reads in contrast to common strategies such as the selection of the longest hit or a random choice among all hits. On benchmark data ChiRA improved the number of correct alignments to the reference up to 3-fold. It is shown that the genes that belong to the common read loci share the same protein families or similar pathways. In published data, ChiRA could detect 3 times more new interactions compared to existing approaches. In addition, ChiRAViz can be used to visualize and filter large chimeric datasets intuitively.Conclusion ChiRA tool suite provides a complete analysis and visualization framework along with ready-to-use Galaxy workflows and tutorials for RNA-RNA interactome and structurome datasets. Common read loci built by ChiRA can rescue multi-mapped reads on paralogous genes without requiring any information on gene relations. We showed that ChiRA is sensitive in detecting new RNA-RNA interactions from published RNA-RNA interactome datasets.

In vivo nuclear RNA structurome reveals RNA-structure regulation of mRNA processing in plants

BackgroundmRNA processing is critical for gene expression. A challenge in regulating mRNA processing is how to recognize the actual mRNA processing sites, such as splice and polyadenylation sites, when the sequence content is insufficient for this purpose. Previous studies suggested that RNA structure affects mRNA processing. However, the regulatory role of RNA structure in mRNA processing remains unclear.ResultsHere, we perform in vivo selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical profiling on Arabidopsis and generate the in vivo nuclear RNA structure landscape. We find that nuclear mRNAs fold differently from cytosolic mRNAs across translation start and stop sites. Notably, we discover a two-nucleotide single-stranded RNA structure feature upstream of 5′ splice sites that is strongly associated with splicing and the selection of alternative 5′ splice sites. The regulatory role of this RNA structure feature is further confirmed by experimental validation. Moreover, we find the single-strandedness of branch sites is also associated with 3′ splice site recognition. We also identify an RNA structure feature comprising two close-by single-stranded regions that is specifically associated with both polyadenylation and alternative polyadenylation events.ConclusionsWe successfully identify pre-mRNA structure features associated with splicing and polyadenylation at whole-genome scale and validate an RNA structure feature which can regulate splicing. Our study unveils a new RNA structure regulatory mechanism for mRNA processing.

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage invivo.

MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in Bacillus subtilis.

RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.

Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions.

The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed ‘Lead-seq’, provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5′-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.

Gene Delivery of RNA Therapeutics for the Treatment of Brain Tumors

The proliferation of glioblastoma multiforme (GBM) is often coupled to the dysregulation, amplification and overexpression of tyrosine kinase receptors (TKRs) including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) among others. TKRs are cell‐surface receptors that bind to their cognate ligand, peptide hormones known as growth factors. Binding of the growth factor ligand stimulates TKRs, activating a signal‐transduction cascade that leads to cell growth, migration and the formation of tumor vasculature. A look at TKRs reveals a general architecture of the receptor. They are comprised of three distinct domains: an extracellular, transmembrane and an intracellular domain. As these receptors are often upregulated in GBM, an RNA Therapy to modulate the expression, inhibit the translation of critical TKR domains or reduce its activation would reduce the functionality of these oncogenic transcripts and proteins in GBM. Our research focuses on the development of AAV gene therapy vectors that encode novel RNA therapeutics to reduce the expression of oncogenic transcripts as well as inhibit their activation in GBM, the most common malignant primary brain tumor in adults. Individuals diagnosed with GBM have a short life expectancy of 12–15 months. The standard care and treatment as well as the developmental pipeline of new therapeutic strategies are often limited by the blood‐brain barrier (BBB), restricting systemically administered therapies from reaching the brain (Hicks et al., 2015). Thus, a novel strategy that circumvents this barrier is necessary to effectively reach the CNS tumor microenvironment. Direct AAV delivery of genes that encode RNA therapeutics at the time of tumor resection would bypass the BBB to deliver a persistent and constant dosage from the inside‐out. With the advancement of nanopore sequencing technology, we are able to read longer transcripts (>200kb). With this technology, we are able to examine the architecture of nascent pre‐mRNA transcripts, detect lower abundant alternatively spliced and polyadenylated isoforms, as well take advantage of RNA structurome analysis to reveal RNA elements within TKRs which are more susceptible to RNA anti‐sense directed therapies. To this end, we have developed a strategy to identify novel targets within oncogenic transcripts and incorporate these therapies directed against these targets into our AAV directed RNA gene therapy platforms. In addition, we use RNA mimics to take advantage of the RNA interference pathway to modulate and reduce expression of oncogenic transcripts. This research has the potential to identify novel RNA elements and alternatively spliced and polyadenylated isoforms of TKRs in health and disease. The ultimate goal of our research project is to build on current strategies to identify even better RNA elements of the EGFR and VEGFR2 transcripts which are structurally available for both microRNA degradation and anti‐sense directed alternative splicing therapy.

RNAdt: An online tutorial and data portal for the RNA structurome era

RNA is not only a passive transporter of genetic information, but also a pivotal player in all domains of life. RNA can regulate gene expression because of its involvement in transcription, mRNA modification and processing, and translation. RNA also possesses other intricate functions such as catalysis, ligand sensing, interaction with biomolecules, response to environment stresses, and information storage. The primary structure of RNA is single stranded, but it always folds into complex secondary and tertiary structures owing to base pairing and effects from the cellular environment. The importance of structure has been increasingly recognized in understanding the myriad functions of RNA. After decades of development, there is a wide range of RNA structure probing techniques. The marriage between structure probing and high-throughput sequencing (HTS) especially enables the measurement of RNA structure on a transcriptomic scale, advancing the advent of the RNA structurome era. Dozens of HTS-associated RNA structure probing methods have been published, so it is urgent to provide a user-friendly and easy-to-use resource for users who are perplexed by selecting the most suitable method for their experiments. Motivated by this demand, we collected currently available HTS-associated RNA structure probing methods and then developed RNAdt (freely accessible at http://www.zhounan.org/rnadt). RNAdt can be used as a web-based tutorial to learn fundamental knowledge of HTS-associated RNA structure probing methods. RNAdt can also be used as a data portal to access HTS data sets from previous RNA structurome studies. At the end of this work, we also provided perspectives on future development of RNA structure probing methods. Our study is expected to facilitate RNA structure probing and ultimately elucidate the connection between RNA structure and biological functions.

DStruct: identifying differentially reactive regions from RNA structurome profiling data

RNA biology is revolutionized by recent developments of diverse high-throughput technologies for transcriptome-wide profiling of molecular RNA structures. RNA structurome profiling data can be used to identify differentially structured regions between groups of samples. Existing methods are limited in scope to specific technologies and/or do not account for biological variation. Here, we present dStruct which is the first broadly applicable method for differential analysis accounting for biological variation in structurome profiling data. dStruct is compatible with diverse profiling technologies, is validated with experimental data and simulations, and outperforms existing methods.

Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis.

Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants.