SARS-CoV-2 RNA stabilizes host mRNAs to elicit immunopathogenesis.

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.

Complementary Alu sequences mediate enhancer-promoter selectivity.

Enhancers determine spatiotemporal gene expression programs by engaging with long-range promoters1-4. However, it remains unknown how enhancers find their cognate promoters. We recently developed a RNA in situ conformation sequencing technology to identify enhancer-promoter connectivity using pairwise interacting enhancer RNAs and promoter-derived noncoding RNAs5,6. Here we apply this technology to generate high-confidence enhancer-promoter RNA interaction maps in six additional cell lines. Using these maps, we discover that 37.9% of the enhancer-promoter RNA interaction sites are overlapped with Alu sequences. These pairwise interacting Alu and non-Alu RNA sequences tend to be complementary and potentially form duplexes. Knockout of Alu elements compromises enhancer-promoter looping, whereas Alu insertion or CRISPR-dCasRx-mediated Alu tethering to unregulated promoter RNAs can create new loops to homologous enhancers. Mapping 535,404 noncoding risk variants back to the enhancer-promoter RNA interaction maps enabled us to construct variant-to-function maps for interpreting their molecular functions, including 15,318 deletions or insertions in 11,677 Alu elements that affect 6,497 protein-coding genes. We further demonstrate that polymorphic Alu insertion at the PTK2 enhancer can promote tumorigenesis. Our study uncovers a principle for determining enhancer-promoter pairing specificity and provides a framework to link noncoding risk variants to their molecular functions.

RIC-seq for global in situ profiling of RNA-RNA spatial interactions.

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.

Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.

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.

RNA-binding protein DDX1 is responsible for fatty acid-mediated repression of insulin translation.

The molecular mechanism in pancreatic ß cells underlying hyperlipidemia and insulin insufficiency remains unclear. Here, we find that the fatty acid-induced decrease in insulin levels occurs due to a decrease in insulin translation. Since regulation at the translational level is generally mediated through RNA-binding proteins, using RNA antisense purification coupled with mass spectrometry, we identify a novel insulin mRNA-binding protein, namely, DDX1, that is sensitive to palmitate treatment. Notably, the knockdown or overexpression of DDX1 affects insulin translation, and the knockdown of DDX1 eliminates the palmitate-induced repression of insulin translation. Molecular mechanism studies show that palmitate treatment causes DDX1 phosphorylation at S295 and dissociates DDX1 from insulin mRNA, thereby leading to the suppression of insulin translation. In addition, DDX1 may interact with the translation initiation factors eIF3A and eIF4B to regulate translation. In high-fat diet mice, the inhibition of insulin translation happens at an early prediabetic stage before the elevation of glucose levels. We speculate that the DDX1-mediated repression of insulin translation worsens the situation of insulin resistance and contributes to the elevation of blood glucose levels in obese animals.

Protocol for profiling virus-to-host RNA-RNA interactions in infected cells by RIC-seq.

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.

Emerging roles of RNA-RNA interactions in transcriptional regulation.

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.

Structural insights reveal the specific recognition of meiRNA by the Mei2 protein.

In the fission yeast Schizosaccharomyces pombe, Mei2, an RNA-binding protein essential for entry into meiosis, regulates meiosis initiation. Mei2 binds to a specific non-coding RNA species, meiRNA, and accumulates at the sme2 gene locus, which encodes meiRNA. Previous research has shown that the Mei2 C-terminal RNA recognition motif (RRM3) physically interacts with the meiRNA 5' region in vitro and stimulates meiosis in vivo. However, the underlying mechanisms still remain elusive. We first employed an in vitro crosslinking and immunoprecipitation sequencing (CLIP-seq) assay and demonstrated a preference for U-rich motifs of meiRNA by Mei2 RRM3. We then solved the crystal structures of Mei2 RRM3 in the apo form and complex with an 8mer RNA fragment, derived from meiRNA, as detected by in vitro CLIP-seq. These results provide structural insights into the Mei2 RRM3-meiRNA complex and reveal that Mei2 RRM3 binds specifically to the UUC(U) sequence. Furthermore, a structure-based Mei2 mutation, Mei2F644A causes defective karyogamy, suggesting an essential role of the RNA-binding ability of Mei2 in regulating meiosis.

Global in situ profiling of RNA-RNA spatial interactions with RIC-seq.

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.

The architecture of the SARS-CoV-2 RNA genome inside virion.

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.

Global profiling of RNA-binding protein target sites by LACE-seq.

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.

Arabidopsis ARGONAUTE 1 Binds Chromatin to Promote Gene Transcription in Response to Hormones and Stresses.

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.

The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes.

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.