RNA Biology最新文献

SARS-CoV-2 is the betacoronavirus causing the COVID-19 pandemic. Although the SARS-CoV-2 genome and transcriptome were reported previously, the function of individual viral proteins is largely unknown. Utilizing biochemical and molecular biology methods, we identified that four SARS-CoV-2 RNA-binding proteins (RBPs) regulate the host RNA metabolism by direct interaction with mature miRNA let-7b revealed by Nuclear Magnetic Resonance spectroscopy (NMR). SARS-CoV-2 RBP Nsp9 primarily binds mature miRNA let-7b, a direct ligand of the Toll-like Receptor 7 (TLR7), one of the potential SARS-CoV-2 therapeutics. Nsp9 suppresses host gene expression possibly by promoting let-7b-mediated silencing of a cellular RNA polymerase, POLR2D. In addition, Nsp9 inhibits extracellular release of let-7b and subsequent antiviral activity via TLR7. These results demonstrate that SARS-CoV-2 hijacks the host RNA metabolism to suppress antiviral responses and to shut down cellular transcription. Our findings of how a natural ligand of TLR7, miRNA let-7b, is suppressed by SARS-CoV-2 RBPs will advance our understanding of COVID-19 and SARS-CoV-2 therapeutics.

CD4+ regulatory T cells (T_REGS) are critical for immune tolerance and the transcription factor Forkhead Box P3 (FOXP3) plays a crucial role in their differentiation and function. Recently, an alternative promoter has been reported for FOXP3, which is active only in T_REGS and could have profound implications for the output of the locus, and therefore, for the functionality of these cells. By direct RNA sequencing we identified multiple novel FOXP3 transcriptional products, including one relatively abundant isoform with an extended 5' UTR that we named 'longFOXP3'. Western blotting, analysis of public mass spectrometry data, and transfection of in vitro transcribed RNA suggested that longFOXP3 is not coding. Furthermore, we show using two distinct RNA single-molecule fluorescence in situ hybridization technologies that transcription from the upstream promoter correlates with decreased levels of FOXP3 at the mRNA and protein levels. Together, we provide compelling evidence that the transcriptional output of the human FOXP3 locus is far more complex than that of the current annotation and warrants a more detailed analysis to identify coding and non-coding transcript isoforms. Furthermore, the alternative promoter may interfere with the activity of the canonical promoter, evoking intragenic transcriptional interference, and in this way, fine-tune the levels of FOXP3 in human T_REGS.

RNA-binding proteins are involved in all steps of gene expression. Their malfunction has important consequences for cell growth through dysregulation of protein synthesis events leading to cancer. Gemin5 is a predominantly cytoplasmic protein involved in spliceosome assembly and gene expression reprogramming. The protein is phosphorylated at multiple sites, although the role of the individual phosphorylated residues remains poorly understood. With the aim to understand the impact of Gemin5 post-translation modifications for RNA-binding, protein synthesis, and therefore cell growth, we have analysed the role of conserved P-residues located in the dimerization domain of the protein in subcellular localization, protein stability, interactome, ribosome binding and translation regulation. We show that the activation of signalling pathways in response to a dsRNA mimic, which leads to phosphorylation of eIF2α, enhanced the intensity of Gemin5 binding to a cognate RNA ligand. In addition, ribosome binding decreased when Ser/Thr 847 and 852-854 are substituted by a non-phosphorylatable residue, consistent with decreased protein stability, and reduced number of associated factors. Similar analyses of phosphomimetic mutants (S847D and STS852-854DDD) suggested conformational changes of the protein structure as the responsible factor for the defective proteins. Moreover, cap-dependent protein synthesis was significantly altered by the triple substitution STS/DDD, pointing towards a role of these residues in protein synthesis regulation.

Antiterminators are essential components of bacterial transcriptional regulation, allowing the control of gene expression in response to fluctuating environmental conditions. Among them, RNA-binding antiterminator proteins play a major role in preventing transcription termination by binding to specific RNA sequences. These RNA-binding antiterminators have been extensively studied for their role in regulating various metabolic pathways. However, their function in modulating the physiology of pathogens requires further investigation. This review focuses on RNA-binding proteins displaying CAT (Co-AntiTerminator) or ANTAR (AmiR and NasR Transcription Antitermination Regulators) domains reported in model bacteria. In particular, their structures, mechanism of action, and target genes will be described. The involvement of the antitermination mechanisms in bacterial pathogenicity is also discussed. This knowledge is crucial for understanding the regulatory mechanisms that control bacterial virulence, and opens up exciting prospects for future research, and potentially new alternative strategies to combat infectious diseases.

More than 4,000 single nucleotide polymorphisms (SNP) variants have been identified in the human ZFP36L2 gene, however only a few have been studied in the context of protein function. The tandem zinc finger domain of ZFP36L2, an RNA binding protein, is the functional domain that binds to its target mRNAs. This protein/RNA interaction triggers mRNA degradation, controlling gene expression. We identified 32 non-synonymous SNPs (nsSNPs) in the tandem zinc finger domain of ZFP36L2 that could have possible deleterious impacts in humans. Using different bioinformatic strategies, we prioritized five among these 32 nsSNPs, namely rs375096815, rs1183688047, rs1214015428, rs1215671792 and rs920398592 to be validated. When we experimentally tested the functionality of these protein variants using gel shift assays, all five (Y154H, R160W, R184C, G204D, and C206F) resulted in a dramatic reduction in RNA binding compared to the WT protein. To understand the mechanistic effect of these variants on the protein/RNA interaction, we employed DUET, DynaMut and PyMOL to investigate structural changes in the protein. Additionally, we conducted Molecular Docking and Molecular Dynamics Simulations to fine tune the active behaviour of this biomolecular system at an atomic level. Our results propose atomic explanations for the impact of each of these five genetic variants identified.

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3' splice site selection. The SF3B1 K700E mutation (lysine to glutamic acid) is common in myelodysplastic syndrome and other blood disorders. SF3B1 K700E mutants utilize novel cryptic 3' splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 cryptic 3' splice junctions with significantly altered use in SF3B1 K700E cells, termed SF3B1-sensitive cryptic 3' splice sites, and 2800 cryptic 3' splice sites used in SF3B1 wild-type, termed SF3B1-resistant. We find that SF3B1-sensitive cryptic 3' splice sites are embedded in extended polypyrimidine tracts. Furthermore, canonical splice sites paired to SF3B1-sensitive cryptic 3' splice sites are significantly weaker than canonical 3' splice sites paired to SF3B1-resistant cryptic 3' splice sites. We test whether SF3B1-sensitive splice sites are structurally different from SF3B1-resistant 3' splice sites using chemical probing. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3' splice sites is similar. However, the magnitude of accessibility differences is less in paired SF3B1-sensitive splice sites than in paired SF3B1-mutant splice sites. Additionally, SF3B1-sensitive splice junctions are more flexible than SF3B1-resistant junctions. Our results suggest that SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3' splice site recognition in the presence of SF3B1 mutation.

Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC - MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m⁶A per RNA molecule. ONT sequencing predicted three m⁶A positions. Employing orthogonal SELECT method, we confirmed m⁶A in two positions A353 and A360, which are highly conserved among CEVd variants. These positions are located in the left terminal region of the CEVd rod-like structure where likely RNA Pol II and and TFIIIA-7ZF bind, thus suggesting potential biological role of methylation in viroid replication.

Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules, which have been implicated in both physiology and human diseases. Most circRNAs are typically generated through backsplicing, where a downstream splice donor is covalently joined to an upstream splice acceptor. Backsplicing is dependent on the spliceosome machinery and is precisely controlled by various cis-elements and trans-factors. In the present review, we summarize the molecular mechanisms of backsplicing regulation as well as their physiological and pathological significance. Additionally, we discuss the strategies to manipulate circRNA expression in vivo and in vitro, aiming to explore the application of circRNA biogenesis in the diagnosis and therapy of human diseases.

Cellular differentiation requires highly coordinated action of all three transcriptional systems to produce rRNAs, mRNAs and various 'short' and 'long' non-coding RNAs by RNA Polymerase I, II and III systems, respectively. RNA Polymerase I catalyzes transcription of about 400 copies of mammalian rDNA genes, generating 18S, 5.8S and 28S rRNA molecules. Lens fiber cell differentiation is a unique process to study transcriptional mechanisms of individual crystallin genes as their very high transcriptional outputs are directly comparable only to globin genes in erythrocytes. Importantly, both terminally differentiated lens fiber cells and mammalian erythrocytes degrade their nuclei through different mechanisms. In lens, the generation of the organelle-free zone (OFZ) includes the degradation of mitochondria, endoplasmic reticulum, Golgi apparatus and nuclei. Here, using RNA fluorescence in situ hybridization (FISH), we evaluated nascent rRNA transcription, located in the nucleoli, during the process of mouse lens fiber cell differentiation. Lens fiber cell nuclei undergo morphological changes including chromatin condensation prior to their denucleation. Remarkably, nascent rRNA transcription persists in all nuclei that are in direct proximity of the OFZ. Additionally, changes in both nuclei and nucleoli shape were evaluated via immunofluorescence detection of fibrillarin, nucleolin, UBF and other proteins. These studies demonstrate for the first time that highly condensed lens fiber cell nuclei have the capacity to support nascent rRNA transcription. Thus, we propose that 'late' production of rRNA molecules and consequently of ribosomes increases crystallin protein synthesis machinery within the mature lens fibers.

Long non-coding RNAs (lncRNAs) exert a significant influence on the occurrence and progression of osteoarthritis (OA). LncRNAs are characterized by their multifunctional nature, capable of regulating the expression, transcription, translation, and structural function of target genes through various mechanisms, spanning epigenetic, transcriptional, post-transcriptional, and post-translational levels. This review examines the mechanisms and functions of lncRNAs in cell proliferation, differentiation, apoptosis, extracellular matrix (ECM) degradation, and inflammatory responses in chondrocytes, synovial cells, and mesenchymal stem cells (MSCs) from mice and humans associated with OA. We emphasize the integral role of lncRNAs in the OA disease process. Conclusively, we present insights into OA treatment from the perspective of targeting lncRNAs, addressing future development prospects and potential clinical applications.