RNA最新文献

Translation elongation defects cause ribosome stalling and activate the integrated stress response (ISR). During the ISR, translation initiation suppression and ribosome runoff drive mRNA condensation into stress granules. However, the effects of partial translation elongation inhibition on stress granules are poorly defined. We demonstrate that intermediate levels of tRNA synthetase inhibitors activate the ISR and cause assembly of stress granules in a parabolic dose-response pattern. These stress granules are limited in size and number due to ribosome association with mRNAs. Assembly of stress granules by intermediate levels of the prolyl-tRNA synthetase inhibitor halofuginone requires the canonical stress granule scaffolding proteins G3BP1/2 and GCN2-mediated ISR activation. We performed a candidate-based comparative analysis of the composition of stress granules induced by intermediate levels of halofuginone or canonical stressors arsenite or thapsigargin. The stress granules induced by halofuginone, arsenite, or thapsigargin harbor polyadenylated RNA and the canonical stress granule proteins PABPC1, G3BP1, and UBAP2L. We observe stress- and transcript- specific differences in the localization of candidate RNA molecules to stress granules. These results demonstrate that partial translation elongation inhibition permits stress granule assembly through the balance of ISR activation and mRNA association with ribosomes, with implications for the stress response associated with amino acid or tRNA deficiency, therapeutic tRNA synthetase inhibition, or diseases associated with tRNA synthetase mutations.

Cell cycle progression requires cells to continually remodel their gene expression programs as they transition through distinct functional states. Although transcriptional and post-translational mechanisms have long dominated our understanding of this regulation, recent work additionally highlights the essential contribution of cell cycle-specific mRNA decay and translational control. Across G1, S, G2, and mitosis, cells dynamically modulate global and transcript-specific mRNA stability and translation to coordinate processes including DNA replication, growth, checkpoint signaling, and chromosome segregation. Mitosis presents a particularly striking challenge: transcription is silenced, necessitating that cells rely on post-transcriptional mechanisms to sustain mitotic functions and preserve viability. In this review, we highlight how these coordinated layers of post-transcriptional regulation collectively contribute to cell cycle control.

The PUF domain of Drosophila Pumilio consists of eight homologous repeats that act in a modular fashion to recognize Nanos Response Elements (NREs) in targeted mRNAs, each repeat interacting with a single base of the NRE. Most of the sequence specificity of binding is thought to be driven by interactions between residues 12 and 16 of each repeat with the edge of the appropriate RNA base. In this report, we investigate the repertoire of amino acids at positions 12 and 16 that are capable of mediating high affinity binding for two of the PUF repeats, R6 and R5. We generate plasmid libraries in which the codons for residues 12 and 16 are randomized, transform these into a suitable yeast strain, and screen for variants that recognize an NRE in three hybrid RNA-binding experiments. We find that the two repeats have very different capabilities. Relatively few amino acid combinations in R6 are functional and these exhibit a limited array of binding specificities; in contrast, approximately 24% of the edge-on amino acid combinations in R5 are functional, and these exhibit a variety of novel specificities. These results support the idea that R6 (in part) defines NRE target sites, while R5 selects among NREs to allow differential regulation in vivo. We also show that R5 binding modules generally cannot be functionally swapped into R6. Thus, although binding of the Pumilio PUF domain is modular, the R6 and R5 modules are not readily interchangeable, consistent with studies of other PUF domains.

Nonsense-Mediated mRNA Decay (NMD) is a translational-dependent mRNA decay pathway that regulates mRNAs and protects cells from the deleterious, truncated protein products of mRNAs with early stop codons. Despite substantial effort, the central biochemical reactions that comprise mRNA decay during NMD remain elusive. Research by our lab and others centers around the observation that NMD target mRNA cleavage by the endonuclease SMG-6 requires the presence of another NMD factor SMG-5, although the molecular basis of SMG-6's requirement for SMG-5 remained elusive. Here we present work to explain the requirement of SMG-5 in SMG-6-mediated mRNA cleavage. We revisit previous observations that SMG-5 contains a catalytically inactive PIN nuclease domain, and we show that although SMG-5 lacks conventional active site residues, the PIN domain of SMG-5 nevertheless contains highly conserved residues that are essential to NMD. We show that Alphafold predicts an interaction between SMG-5 and SMG-6 PIN domains, an interaction that we substantiate via in vitro pulldowns. We use the in silico models to design point mutations that perturb-and restore-NMD function in C. elegans via a compensatory salt bridge flip. Altogether, our data support the idea that SMG-5 and SMG-6 interact to form a functional complex, and we suggest molecular roles for the overlooked SMG-5 PIN domain in SMG-6-mediated mRNA cleavage.

The 3'-5' RNA polymerase family consists of eukaryotic tRNAHis guanylyltransferase (Thg1) and Thg1 homologs known as Thg1-like proteins (TLPs) that exist in all three domains of life. Thg1 catalyzes an essential reaction adding a G-1 nucleotide to the 5' end of the tRNAHis, forming an identity element for tRNA aminoacylation. All TLPs studied, except Dictyostelium discoideum (Ddi) TLP2, perform in vitro Watson-Crick (WC) dependent addition of multiple nucleotides to repair truncated tRNA. DdiTLP2 has similar activity to Thg1, adding G-1 to mt-tRNAHis, but shares other biochemical properties with other TLPs, including a restriction to making WC base pairs during this reaction. We identified two regions in DdiTLP2 that lacked residues that are conserved in other Thg1/TLP enzymes. DdiTLP2 variants in both regions abolish enzymatic activity of DdiTLP2, indicating these regions are important for DdiTLP2 catalysis. Changing DdiThg1 D150 to the corresponding arginine found in DdiTLP2 causes an unexpected reversal of this enzyme's specificity, with a loss of its ability to incorporate a non-WC base paired G-1 to its physiological substrate, while gaining the ability to add WC base paired G-1 to mt-tRNAHis. Biochemical study of other changes to D150, combined with structural models, suggest a previously unknown role for D150 in controlling substrate specificity at the adenylation step by providing a checkpoint for correct setup of a WC base pair in the active site. Thg1 also appears to have adapted the role of the ancestral D150 residue for a second function, promoting non-WC nucleotide addition to its eukaryotic tRNAHis substrate.

Enhancer RNAs (eRNAs) are best known for their role in transcriptional regulation, where they facilitate enhancer-promoter communication and chromatin remodelling. Yet growing evidence suggests that their function may extend beyond the nucleus. Here, we systematically characterise the decay kinetics of eRNAs across human cell types using time-resolved transcriptomics and kinetic modelling. While most eRNAs undergo canonical exponential decay, a subset displays non-linear dynamics, suggesting context-dependent degradation mechanisms. Perturbation of core decay regulators, including components of the m⁶A and CCR4-NOT pathways, reveals that eRNA stability is modulated by a patchwork of pathways governing mRNA turnover. Integrating transcriptome-wide ribosome profiling, RNA-Seq, and half-life data, we identify eRNAs associated with changes in mRNA stability and translation efficiency of their target protein-coding transcripts. Functional validation of one such eRNA, en4528, shows it regulates CDKN2C mRNA independently of transcription and impacts cell migration. These findings redefine the regulatory scope of eRNAs, positioning them as active participants in post-transcriptional gene control and cellular behaviour. The resulting decay profiles and regulatory annotations have been incorporated into the eRNAkit database, available at https://github.com/AneneLab/eRNAkit, enhancing its capacity for integrative systems-level analysis of eRNA function.

Internal ribosome entry sites (IRESs) are RNA sequences that facilitate cap- and end-independent translation initiation in eukaryotes. Type IV IRESs, which include the hepatitis C virus IRES, directly bind the 40S ribosomal subunit and require only a subset of canonical initiation factors to function. As the full extent of diversity and species distribution of Type IV IRESs was unknown, we sought to identify and classify the architectural variation of all members. Using a secondary structure homology-based search method, we identified 163 putative Type IV IRESs from viruses with diverse hosts and phylogeny, including the first example in a double stranded viral genome. Clustering analysis based on the presence and overall size of secondary structure elements yielded three distinct groups, differentiated by substantial expansions and deletions. Chemical probing of representative IRES RNAs from each cluster confirmed predicted secondary structures. Subsequent in vitro translation assays suggested that structural differences produce functional variation. Our findings reveal distinct structural adaptations and patterns within the Type IV IRESs that may influence IRES function and mechanism.

The concerted action of regulatory RNA and RNA binding proteins (RBPs) provide cells with highly versatile and transient tools to fine tune gene expression in a broad variety of cellular systems (Unfried and Ulitsky 2022, Hentze et al. 2018, Suzuki et al. 2018). In this work, we explore the function of a specific interaction between PTBP1 and the cytoplasmic long non-coding RNA (lncRNA) CyCoNP, highly expressed in neural progenitors (Desideri et al. 2024), in which the RBP regulates the abundance of the lncRNA by a miRNA-mediated mechanism. PTBP1 is a well-known splicing regulator, although limited and peculiar examples of its involvement in other cellular processes, such as IRES-dependent translation and miRNA recognition of target RNAs, have been described (Dorn et al. 2023, Kim et al. 2021). We have recently characterized CyCoNP lncRNA as a regulator of NCAM1, which acts through a mechanism that involves direct RNA-RNA interaction with NCAM1 mRNA, balancing the availability and the localization of miR-4492 in its vicinity (Desideri et al. 2024). Here we expand the repertoire of molecular players acting in this circuitry by describing a direct interaction between PTBP1 and CyCoNP lncRNA. Through endogenous RNA purification, protein immunoprecipitation and exploiting CyCoNP mutant constructs we found that PTBP1, when interacting with CyCoNP, hampers miR-4492 binding to the lncRNA and in turn impedes its regulation on NCAM1 mRNA. This work aims to expand the biochemical characterization of regulatory networks relying on RBPs and their cognate target RNAs, highlighting the relevance of the analysis of the subcellular environment for each case of study.

DDX3X is a human DEAD-Box RNA helicase with multiple functions in RNA metabolism. Previous studies have suggested that DDX3X is an important pro-viral host factor for numerous RNA viruses, including HIV, HCV, and SARS-CoV-2, and may be targetable with inhibitors such as RK-33 for therapeutic benefit. In exploring the role of DDX3X and its homolog DDX3Y in coronavirus replication, we found that the DDX3X inhibitor RK-33 inhibits propagation of the OC43 coronavirus through a DDX3X/DDX3Y-independent mechanism. Knockdowns of DDX3X or DDX3X and DDX3Y had little effect on OC43 growth in multiple cell lines, yet RK-33 treatment potently reduced OC43 replication in the presence or absence of DDX3 proteins. We observed that RK-33 stimulates the integrated stress response independently of DDX3 proteins to cause stress granule formation, although this is not the primary mechanism by which RK-33 suppresses OC43. Together, our results show that DDX3 proteins are likely not a general pro-coronaviral host factor, and caution should be used in interpreting results with RK-33 given its off-target activity.

Almost all protein-coding and long noncoding genes that are transcribed by RNA polymerase II employ cleavage and polyadenylation (CPA) for 3' end maturation of their nascent RNAs. More than 70% of human mRNA genes display alternative polyadenylation (APA), resulting in expression of isoforms using different polyA sites (PAS). APA isoforms often have distinct mRNA metabolism and/or contain variable coding sequences. PAS mutations and genetic variations have been implicated in a growing number of human pathological conditions, underscoring the importance of PAS usage or selection for proper gene expression. Here we review approaches that modulate the usage of specific PAS using antisense- and CRISPR-based methods. We discuss applications of these strategies in the context of human diseases. We provide our perspectives on current challenges and future directions to advance PAS modulation in APA studies and development of related therapeutics.