RNA最新文献

One of the striking characteristics of eukaryotic genomes is the presence of three types of introns: spliceosomal introns, tRNA introns, and a unique intron in the XBP1 mRNA. Exceptional eukaryotic genomes that lack spliceosomal or XBP1 introns have been described. However, tRNA introns and the tRNA endonuclease that is required for their splicing are thought to be universal in eukaryotes. The introns in three tRNAs are widely conserved across Metazoa: Tyr-GUA, Ile-UAU, and Leu-CAA. This study shows that some nematode species have lost the introns in Tyr-GUA and Ile-UAU tRNAs, and one species, Levipalatum texanum, completely lacks tRNA introns. The loss of the intron from Leu-CAA tRNA is accompanied by an unusual A-C mismatched base pair in the anticodon stem-loop and a triplication of a tRNA deaminase that could potentially restore base-pairing. These changes may be an adaptation to the loss of the intron. L. texanum also lacks the tRNA endonuclease, one of two enzymes required for tRNA splicing. The other key enzyme in tRNA splicing, tRNA ligase, is bifunctional and is also required for XBP1 mRNA splicing. L. texanum retains tRNA ligase and the XBP1 intron. This eukaryote without tRNA introns has the potential to be a valuable tool for disentangling the functions of tRNA splicing, XBP1 splicing, and tRNA modification enzymes and is the only animal known to have lost one of the three intron types.

RNA G-quadruplexes (rG4s) are unusual RNA secondary structures formed by stacking arrays of guanine tetrads. Although thousands of potential rG4-forming motifs occur throughout the mammalian transcriptome, many single-stranded RNA (ssRNA) viruses are thought to be depleted of rG4-forming sequences. Using in silico methods, we examine rG4-forming potential in single-stranded RNA (ssRNA) viruses and observe that, while canonical rG4 motifs are depleted, noncanonical rG4 motifs occur at comparable or higher frequencies relative to the mammalian transcriptome. We ask if the noncanonical rG4's can be leveraged to block viral replication and control infection using OC43, the coronavirus believed to be responsible for the 1889 "Russian flu" pandemic. Profiling with "d-rG4-seq" confirms a dearth of folded rG4 in the OC43 RNA genome during natural infection. Intriguingly, rG4 ligands induce synthetic rG4 structures of a noncanonical nature. Significantly, induced rG4 inhibits viral replication and reduces infectivity. We show that the rG4 ligands act by disrupting the unique pattern of OC43 "discontinuous transcription." Thus, rG4-targeting compounds present a potential therapeutic approach for targeting ssRNA viruses.

The RNA-binding protein CSDE1 is a key regulator of mRNA stability and translation in a broad spectrum of biological processes. We have previously shown that CSDE1 functions as an oncoprotein promoting invasion and metastasis in melanoma, whereas it behaves as a tumor suppressor promoting cellular senescence in squamous cell carcinoma. The reasons underlying these context-specific behaviors are unknown. To identify melanoma-specific vulnerabilities, we have compared CSDE1 protein isoforms and post-translational modifications in melanoma cells, keratinocytes, and melanocytic cells of different tumorigenic potential. By combining long-read Nanopore sequencing with two-dimensional gel electrophoresis and transcriptome analysis, we identify one major isoform expressed in melanoma cells and patient samples. This isoform is phosphorylated early during cellular transformation, correlating with changes in its subcellular localization. We provide extensive interactome analysis of mammalian CSDE1, showing increased interactions with ribosomes in melanoma cells compared to healthy melanocytes. Importantly, interactions of CSDE1 with the ribosome are promoted by CSDE1 phosphorylation. Our data uncover a specific feature of melanoma cells that could be harnessed for therapeutic intervention.

RNA modifications, especially m⁶A in human mRNA, are believed to be dynamically regulated through RNA writers and erasers. The key eraser of m⁶A is ALKBH5 with its function well proven in vitro, while in vivo evidence is lacking. Here, we set out to exploit nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) in a pulse chase setup to study the in vivo function of ALKBH5 on human RNAs. For this, we purified poly(A) from whole-cell total RNA and found that steady-state m⁶A levels and turnover dynamics were nearly identical between WT and ALKBH5 KO, despite clear evidence of robust RNA turnover within an 8 h labeling period. To assess whether ALKBH5 might act in a compartment-specific manner, we used an advanced subcellular fractionation strategy, allowing for the isolation of chromatin-associated, nucleoplasmic, and cytoplasmic RNA. These analyses confirmed that m⁶A accumulates during transcript maturation, with levels peaking in nuclear fractions and decreasing following export to the cytoplasm, supporting the thesis that m⁶A is a dynamic modification. Notably, however, spatial and temporal profiles of m⁶A distribution and decay were unaffected by ALKBH5 KO. Even in chromatin-associated and nucleolar mRNA, where cotranscriptional modification and potential demethylation would be most plausible, m⁶A dynamics remained indistinguishable between WT and KO cells in the NAIL-MS context. We thus conclude that ALKBH5 has no major role in global mRNA m⁶A turnover in HEK 293T cells grown under optimal conditions.

Heat shock proteins have been increasingly identified in RNA-interactomes, suggesting potential roles beyond their canonical functions. Among those, the cancer-linked chaperone TRAP1 has been mainly characterized for its regulatory role on respiratory complex activity and protein synthesis, while its specific function as an RNA-binding protein (RBP) remains unclear. In this study, we confirmed the RNA-binding activity of TRAP1 in living cells using both protein- and RNA-centric approaches and demonstrated that multiple TRAP1 regions cooperate in such binding. Enhanced cross-linking and immunoprecipitation (eCLIP) in high-grade serous ovarian cancer cells revealed that TRAP1 primarily binds cytosolic protein-coding genes, with the majority coding for splicing-related factors. Notably, among TRAP1 most significantly bound transcripts, we identified the splicing factor LUC7L3, a U1 snRNP component involved in cell proliferation. We confirmed TRAP1 binding to the Luc7l3 transcript by RIP-qPCR and showed that TRAP1 promotes Luc7l3 mRNA translation. Furthermore, we demonstrated that TRAP1 enhances ovarian cancer cell proliferation through LUC7L3 translational regulation. In summary, our findings provide the first comprehensive characterization of TRAP1 as an RBP and identify a critical target for ovarian cancer cell proliferation, offering new insights into its multifaceted roles in tumor biology.

Sexually reproducing organisms make haploid gametes-oocytes and spermatocytes-that combine during fertilization to make an embryo. While both gametes contain similar DNA content, oocytes contain the bulk of the cytoplasm including maternally supplied mRNAs and proteins required prior to zygotic gene activation. RNA-binding proteins are key regulators of these maternal transcripts. In Caenorhabditis elegans, the tandem zinc finger proteins OMA-1 and OMA-2 are required for fertilization. Here, we show that OMA-1 RNA-binding activity requires a short basic region immediately upstream of the canonical tandem zinc finger domain. Mutation of this region in animals produces a phenotype distinct from a genetic null. Oocytes can be fertilized, but fail to form an intact chitin eggshell, frequently fragment in utero, and arrest prior to morphogenesis. Our results identify a critical region outside of the canonical RNA-binding domain required for both RNA-binding activity as well as revealing a new role for OMA-1 during the oocyte-to-embryo transition.

CpG dinucleotides are underrepresented in the genomes of most RNA viruses. Synonymously increasing CpG content of a range of RNA virus genomes reliably causes replication defects due to the recognition of CpG motifs in RNA by cellular zinc-finger antiviral protein (ZAP). Prior to the discovery of ZAP as a CpG sensor, we described an engineered influenza A virus (IAV) enriched for CpGs in segment 5 that displays the expected replication defects. However, we report here that this CpG-high ("CpGH") mutant is not attenuated by ZAP. Instead, a pair of compensatory nucleotide changes, resulting in a stretch of eight consecutive adenosines (8A), were found to be responsible. Viral polymerase slippage occurs at this site, resulting in the production of aberrant peptides and type I interferon induction. When the nucleotides in either one of these two positions were restored to wild-type sequence, no viral attenuation was seen, despite the 86 extra CpGs encoded by this virus. Introduction of these two adenosines into wild-type virus (thereby introducing the 8A tract) resulted in viral attenuation, polymerase slippage, aberrant peptide production and type I interferon induction. That a single nucleotide change can offset the growth defects in a virus designed to have a formidable barrier to wild-type reversion highlights the importance of understanding the processes underlying viral attenuation. Poly(A) tracts are a correlate for the emergence of polybasic cleavage sites in avian IAV hemagglutinins to produce highly pathogenic strains. These results thereby uncover possible insights into the intermediary events of this important evolutionary process.

In clinical uses, RNA must maintain its integrity in serum that contains ribonucleases (RNases), especially RNase 1, which is a human homolog of RNase A. These omnipresent enzymes catalyze the cleavage of the P-O^5″ bond on the 3' side of pyrimidine residues. Pseudouridine (Ψ) is the most abundant modified nucleoside in natural RNA. The substitution of uridine (U) with Ψ or N ¹-methylpseudouridine (m¹Ψ) reduces the immunogenicity of mRNA and increases ribosomal translation, and these modified nucleosides are key components of RNA-based vaccines. Here, we assessed the ability of RNase A and RNase 1 to catalyze the cleavage of the P-O^5″ bond on the 3' side of Ψ and m¹Ψ. We find that these enzymes catalyze the cleavage of UpA up to 10-fold more efficiently than the cleavage of ΨpA or m¹ΨpA. X-ray crystallography of enzyme-bound nucleoside 2',3'-cyclic vanadate complexes and molecular dynamics simulations of enzyme·dinucleotide complexes show that U, Ψ, and m¹Ψ bind to RNase A and RNase 1 in a similar manner. Quantum chemistry calculations suggested that the higher reactivity of UpA is intrinsic, arising from an inductive effect that decreases the pK _a of the 2'-hydroxy group of U and enhances its nucleophilicity toward the P-O^5″ bond. Experimentally, we found that UpA does indeed undergo spontaneous hydrolysis faster than does m¹ΨpA. Our findings reveal a new role for natural pseudouridine residues and inform the continuing development of RNA-based vaccines and therapeutic agents.

The fission yeast phosphate acquisition (PHO) regulon is repressed under phosphate-replete conditions by upstream lncRNA-mediated transcriptional interference. Inositol-1-pyrophosphates control phosphate homeostasis via their action as agonists of precocious PHO lncRNA 3'-processing/termination. Inositol pyrophosphatase-inactivating mutations that increase inositol-1-pyrophosphates elicit derepression of the PHO genes and a severe growth defect in YES medium. Previous studies demonstrated suppression of inositol pyrophosphate toxicosis by targeted deletion or loss-of-function mutations in the nonessential Ssu72, Ppn1, Swd22, and Ctf1 subunits of the fission yeast cleavage and polyadenylation factor (CPF) complex. Here we conducted a selection for spontaneous mutations that suppress the precocious PHO lncRNA termination underlying the sickness of asp1-STF pyrophosphatase mutants. We thereby recovered and characterized novel hypomorphic missense mutations in five essential CPF subunits: Ysh1 (the cleavage endonuclease), Pta1 (an armadillo/HEAT-repeat protein), Pfs2 (a WD repeat protein), Cft1 (a WD repeat protein), and Msi2 (a tandem RRM RNA-binding protein). The suppressor screen also yielded an intron branchpoint mutation in the gene encoding essential CPF subunit Iss1. In addition, we found that asp1-STF toxicosis was suppressed by a missense mutation in the active site of Pla1, the essential poly(A) polymerase subunit of CPF. Genetic crosses revealed a hierarchy of mutational synergies between the essential CPF subunits, the inessential CPF subunits, termination factor Rhn1, the Thr4 "letter" of the RNA polymerase II CTD code, and the Asp1 kinase that synthesizes inositol-1-pyrophosphates. The synthetic lethality of msi2-G252E with ctf1Δ, swd22Δ, ppn1Δ, ssu72-C13S, rpb1-CTD-T4A, and asp1Δ establishes Msi2 as a central agent of 3'-processing/termination, functioning in parallel to inositol-1-pyrophosphates.