SARS-CoV-2 carries the largest single-stranded RNA genome and is the causal pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome is folded in the virion remains unknown. To fill ...the knowledge gap and facilitate structure-based drug development, we develop a virion RNA in situ conformation sequencing technology, named vRIC-seq, for probing viral RNA genome structure unbiasedly. Using vRIC-seq data, we reconstruct the tertiary structure of the SARS-CoV-2 genome and reveal a surprisingly "unentangled globule" conformation. We uncover many long-range duplexes and higher-order junctions, both of which are under purifying selections and contribute to the sequential package of the SARS-CoV-2 genome. Unexpectedly, the D614G and the other two accompanying mutations may remodel duplexes into more stable forms. Lastly, the structure-guided design of potent small interfering RNAs can obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a framework for studying the genome structure, function, and dynamics of emerging deadly RNA viruses.
Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions ...in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.
Conventional RNA interference (RNAi) pathways suppress eukaryotic gene expression at the transcriptional or post-transcriptional level. At the core of RNAi are small RNAs (sRNAs) and effector ...Argonaute (AGO) proteins. Arabidopsis AGO1 is known to bind microRNAs (miRNAs) and post-transcriptionally repress target genes in the cytoplasm. Here, we report that AGO1 also binds to the chromatin of active genes and promotes their transcription. We show that sRNAs and SWI/SNF complexes associate with nuclear AGO1 and are required for AGO1 binding to chromatin. Moreover, we show that various stimuli, including plant hormones and stresses, specifically trigger AGO1 binding to stimulus-responsive genes. Finally, we show that AGO1 facilitates the induction of genes in jasmonate (JA) signaling pathways and the activation of JA responses. Our findings suggest that, by binding and facilitating the expression of stimuli-specific genes, AGO1 may regulate diverse signaling pathways and associated biological processes.
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•AGO1 binds to the chromatin of transcribed genes•AGO1 binding to chromatin requires small RNAs and SWI/SNF•AGO1 promotes the transcription of AGO1-bound genes•Hormone and stress stimuli trigger AGO1 binding to stimulus-responsive genes
Argonaute (AGO) proteins are well known to bind small RNAs and direct post-transcriptional gene silencing. Liu et al. uncover an unsuspected role for Arabidopsis AGO1 in direct chromatin binding to promote gene transcription in responses to stimuli, including hormones and stresses. AGO1 binding requires small RNAs and the SWI/SNF chromatin-remodeling complexes.
RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance ...cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of complementary DNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. Unexpectedly, transcriptomics and proteomics analysis of Ago2
oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP-binding sites in low-input cells opens the door to studying the roles of RBPs in embryonic development and reproductive diseases.
Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ ...conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix all the protein-mediated RNA-RNA interactions in situ. After cell permeabilization and micrococcal nuclease digestion, the proximally interacting RNAs are 3' end-labeled with pCp-biotin and subsequently ligated using T4 RNA ligase. The chimeric RNAs are then enriched and converted into libraries for paired-end sequencing. After deep sequencing, computational analysis yields interaction strength scores for every base on proximally interacting RNAs in the starting populations. The whole experimental procedure is designed to be completed within 6 d, followed by an additional 8 d for computational analysis. RIC-seq technology can unbiasedly detect intra- and intermolecular RNA-RNA interactions, thereby rendering it useful for reconstructing RNA higher-order structures and identifying direct noncoding RNA targets.
Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we ...present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts.
For complete details on the use and execution of this protocol, please refer to Zhao et al.1
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•RIC-seq for mapping virus-to-host in situ RNA-RNA interactions•Step-by-step protocol for constructing RIC-seq libraries•Pipelines of RIC-seq data analysis•Identification of high-confidence virus-to-host RNA-RNA interactions
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts.
Activation-induced cytidine deaminase (AID) mediates class switching by binding to a small fraction of single-stranded DNA (ssDNA) to diversify the antibody repertoire. The precise mechanism for ...highly selective AID targeting in the genome has remained elusive. Here, we report an RNA-binding protein, ROD1 (also known as PTBP3), that is both required and sufficient to define AID-binding sites genome-wide in activated B cells. ROD1 interacts with AID via an ultraconserved loop, which proves to be critical for the recruitment of AID to ssDNA using bi-directionally transcribed nascent RNAs as stepping stones. Strikingly, AID-specific mutations identified in human patients with hyper-IgM syndrome type 2 (HIGM2) completely disrupt the AID interacting surface with ROD1, thereby abolishing the recruitment of AID to immunoglobulin (Ig) loci. Together, our results suggest that bi-directionally transcribed RNA traps the RNA-binding protein ROD1, which serves as a guiding system for AID to load onto specific genomic loci to induce DNA rearrangement during immune responses.
Pervasive transcription of the human genome generates a massive amount of noncoding RNAs (ncRNAs) that lack protein‐coding potential but play crucial roles in development, differentiation, and ...tumorigenesis. To achieve these biological functions, ncRNAs must first fold into intricate structures via intramolecular RNA–RNA interactions (RRIs) and then interact with different RNA substrates via intermolecular RRIs. RRIs are usually facilitated, stabilized, or mediated by RNA‐binding proteins. With this guiding principle, several protein‐based high‐throughput methods have been developed for unbiased mapping of defined or all RNA‐binding protein‐mediated RRIs in various species and cell lines. In addition, some chemical‐based approaches are also powerful to detect RRIs globally based on the fact that RNA duplex can be cross‐linked by psoralen or its derivative 4′‐aminomethyltrioxsalen. These efforts have significantly expanded our understanding of RRIs in determining the specificity and variability of gene regulation. Here, we review the current knowledge of the regulatory roles of RRI, focusing on their emerging roles in transcriptional regulation and nuclear body formation.
This article is categorized under:
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
RNA–RNA interactions determine the shape of various RNA molecules and play crucial roles in posttranscriptional and especially transcriptional gene regulation.
ABSTRACT
In addition to preventing insect metamorphosis, juvenile hormone (JH) is known to stimulate aspects of insect reproduction. However, the molecular mechanisms of JH action in insect ...reproduction remain largely unknown. By reanalyzing the transcriptomic data from adults and other developmental stages of the migratory locust Locusta migratoria, we identified a gene coding for Kazal‐type protease inhibitor, previously named Greglin. Greglin is specifically expressed in adult females and most abundant in the fat body and ovaries. Interestingly, Greglin is among the top 3 of highly expressed genes in adult female locusts, after 2 vitellogenin (Vg) genes. Greglin is induced by JH and expressed at remarkably high levels in the vitellogenic stage. Knockdown of Greglin in adult female locusts results in accelerated degradation of serine protease substrate and significantly reduced levels of Greglin protein in hemolymph and ovaries. The consequent phenotypes include blocked oocyte maturation, arrested ovarian growth and shrunken follicular epithelium, as well as declines in egg number and hatchability. The data provide the first evidence, to our knowledge, that JH‐dependent Greglin is involved in locust vitellogenesis and oocyte maturation likely by protecting vitellogenesis and other forms of yolk precursors from proteolysis. The result offers new insights into the regulation of JH and function of protease inhibitors in insect vitellogenesis, oocyte maturation and fecundity.—Guo, W., Wu, Z., Yang, L., Cai, Z., Zhao, L., Zhou, S. Juvenile hormone–dependent Kazal‐type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production. FASEB J. 33, 917–927 (2019). www.fasebj.org
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