Genome-Wide Analysis of RNA Secondary Structure.

Single-stranded RNA molecules fold into extraordinarily complicated secondary and tertiary structures as a result of intramolecular base pairing. In vivo, these RNA structures are not static. Instead, they are remodeled in response to changes in the prevailing physicochemical environment of the cell and as a result of intermolecular base pairing and interactions with RNA-binding proteins. Remarkable technical advances now allow us to probe RNA secondary structure at single-nucleotide resolution and genome-wide, both in vitro and in vivo. These data sets provide new glimpses into the RNA universe. Analyses of RNA structuromes in HIV, yeast, Arabidopsis, and mammalian cells and tissues have revealed regulatory effects of RNA structure on messenger RNA (mRNA) polyadenylation, splicing, translation, and turnover. Application of new methods for genome-wide identification of mRNA modifications, particularly methylation and pseudouridylation, has shown that the RNA "epitranscriptome" both influences and is influenced by RNA structure. In this review, we describe newly developed genome-wide RNA structure-probing methods and synthesize the information emerging from their application.

The RNA structurome in the asexual blood stages of malaria pathogen plasmodium falciparum.

Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite's asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5' UTR, CDS and 3' UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs. Abbreviations: CDS: Coding Sequence; DNA: Deoxyribonucleic Acid; dsRNA: double-stranded RNA; IDC: Intra-erythrocytic Developmental Cycle (IDC); m6A: N6-methyladenosine; mRNA: Messenger RNA; ncRNA: Non-coding RNA; RBC: Red Blood cells; RBP: RNA-Binding Protein; REC: Relative Expression Counts; RNA-seq: RNA-sequencing; RNA: Ribonucleic Acid; RNP: Ribonucleoprotein; RPKM: Reads Per Kilobase of transcript Per Million; rRNA: Ribosomal RNA 16. RUFs: RNAs of Unknown Function; SHAPE: Selective 2'-hydroxyl acylation analysed by primer extension; snoRNA: Small Nucleolar RNA; snRNA: Small Nuclear RNA; SRP-RNA: Signal Recognition Particle RNA; ssRNA: (Single-stranded RNA); TE: Translation Efficiency; tRNA: transfer RNA; UTR: Untranslated Region.

The RNA structurome: transcriptome-wide structure probing with next-generation sequencing.

RNA folds into intricate structures that enable its pivotal roles in biology, ranging from regulation of gene expression to ligand sensing and enzymatic functions. Therefore, elucidating RNA structure can provide profound insights into living systems. A recent marriage between in vivo RNA structure probing and next-generation sequencing (NGS) has revolutionized the RNA field by enabling transcriptome-wide structure determination in vivo, which has been applied to date to human cells, yeast cells, and Arabidopsis seedlings. Analysis of resultant in vivo 'RNA structuromes' provides new and important information regarding myriad cellular processes, including control of translation, alternative splicing, alternative polyadenylation, energy-dependent unfolding of mRNA, and effects of proteins on RNA structure. An emerging view suggests potential links between RNA structure and stress and disease physiology across the tree of life. As we discuss here, these exciting findings open new frontiers into RNA biology, genome biology, and beyond.

Insight into HOTAIR structural features and functions as landing pads for transcription regulation proteins.

LncRNAs fulfill a wide range of regulatory functions at almost every process of gene expression. While derived from secondary structural features, lncRNAs may function as landing pads for transcription factors (TFs). In this paper, we detected the global structural landscape of 20,338 lncRNAs by utilizing a free energy minimization (MFE) algorithm, and identified the interactions between lncRNAs and TFs to analyze molecular association induced by the lncRNA structure. The accessibility analysis of full sequences as well as potential TF-binding fragments shows a large percentage of structural flanking sequence around the TF binding sites. This investigations paid great attention to the high-order architecture of HOTAIR lncRNA, and identified two coincident modular domains covering fragments 171-410bp and 811-1520bp via RNA-TF association predicting and in-silico computation mining. Then, the structural domains were implied potential landing pads to recruit regulatory proteins (13 TFs) and mediated coordinate regulation of transcription. Pathways and diseases enrichment analysis illustrated that the interacted TFs are significantly Pan-cancer relevant which is consistent with the known function of HOTAIR. Overall, the in-depth understanding of HOTAIR structure provides the first glimpse of coordinate regulation driven by modular features. The detailed architectural context could yield broad biological insights and provides a framework for comprehending lncRNA structure-function interrelationships.

Dawn of the in vivo RNA structurome and interactome.

RNA is one of the most fascinating biomolecules in living systems given its structural versatility to fold into elaborate architectures for important biological functions such as gene regulation, catalysis, and information storage. Knowledge of RNA structures and interactions can provide deep insights into their functional roles in vivo For decades, RNA structural studies have been conducted on a transcript-by-transcript basis. The advent of next-generation sequencing (NGS) has enabled the development of transcriptome-wide structural probing methods to profile the global landscape of RNA structures and interactions, also known as the RNA structurome and interactome, which transformed our understanding of the RNA structure-function relationship on a transcriptomic scale. In this review, molecular tools and NGS methods used for RNA structure probing are presented, novel insights uncovered by RNA structurome and interactome studies are highlighted, and perspectives on current challenges and potential future directions are discussed. A more complete understanding of the RNA structures and interactions in vivo will help illuminate the novel roles of RNA in gene regulation, development, and diseases.

An ultra low-input method for global RNA structure probing uncovers Regnase-1-mediated regulation in macrophages.

To enable diverse functions and precise regulation, an RNA sequence often folds into complex yet distinct structures in different cellular states. Probing RNA in its native environment is essential to uncovering RNA structures of biological contexts. However, current methods generally require large amounts of input RNA and are challenging for physiologically relevant use. Here, we report smartSHAPE, a new RNA structure probing method that requires very low amounts of RNA input due to the largely reduced artefact of probing signals and increased efficiency of library construction. Using smartSHAPE, we showcased the profiling of the RNA structure landscape of mouse intestinal macrophages upon inflammation, and provided evidence that RNA conformational changes regulate immune responses. These results demonstrate that smartSHAPE can greatly expand the scope of RNA structure-based investigations in practical biological systems, and also provide a research paradigm for the study of post-transcriptional regulation.

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.

Profiling of RNA Structure at Single-Nucleotide Resolution Using nextPARS.

RNA molecules play important roles in almost every cellular process, and their functions are mediated by their sequence and structure. Determining the secondary structure of RNAs is central to understanding RNA function and evolution. RNA structure probing techniques coupled to high-throughput sequencing allow determining structural features of RNA molecules at transcriptome-wide scales. Our group recently developed a novel Illumina-based implementation of in vitro parallel probing of RNA structures called nextPARS.Here, we describe a protocol for the computation of the nextPARS scores and their use to obtain the structural profile (single- or double-stranded state) of an RNA sequence at single-nucleotide resolution.

New Era of Studying RNA Secondary Structure and Its Influence on Gene Regulation in Plants.

The dynamic structure of RNA plays a central role in post-transcriptional regulation of gene expression such as RNA maturation, degradation, and translation. With the rise of next-generation sequencing, the study of RNA structure has been transformed from in vitro low-throughput RNA structure probing methods to in vivo high-throughput RNA structure profiling. The development of these methods enables incremental studies on the function of RNA structure to be performed, revealing new insights of novel regulatory mechanisms of RNA structure in plants. Genome-wide scale RNA structure profiling allows us to investigate general RNA structural features over 10s of 1000s of mRNAs and to compare RNA structuromes between plant species. Here, we provide a comprehensive and up-to-date overview of: (i) RNA structure probing methods; (ii) the biological functions of RNA structure; (iii) genome-wide RNA structural features corresponding to their regulatory mechanisms; and (iv) RNA structurome evolution in plants.

RNA Regulations and Functions Decoded by Transcriptome-wide RNA Structure Probing.

RNA folds into intricate structures that are crucial for its functions and regulations. To date, a multitude of approaches for probing structures of the whole transcriptome, i.e., RNA structuromes, have been developed. Applications of these approaches to different cell lines and tissues have generated a rich resource for the study of RNA structure-function relationships at a systems biology level. In this review, we first introduce the designs of these methods and their applications to study different RNA structuromes. We emphasize their technological differences especially their unique advantages and caveats. We then summarize the structural insights in RNA functions and regulations obtained from the studies of RNA structuromes. And finally, we propose potential directions for future improvements and studies.