RNA最新文献

Proteins have traditionally been understood through their tertiary structures, with well-defined conformations considered essential for biological function. This classical structure-function paradigm implies that proteins with high intrinsic disorder would be less critical for cellular survival. Recent discoveries have suggested that some intrinsically disordered proteins or even fully disordered proteins without any apparent tertiary structures are essential. However, the biological significance of such disordered proteins is not comprehensively understood. Here, using genome-wide CRISPR screening, we demonstrated that highly or fully disordered proteins show comparable essentiality to well-folded proteins. We found that the proportion of essential proteins is comparable across proteins of varying disorder levels, although structured proteins are more prevalent among essential genes. Focusing on FAM32A, one of the essential, fully disordered proteins identified in our screen, we show that its depletion leads to increased intron retention and downregulation of many other essential genes. These findings reshape our understanding of the structure-function paradigm, highlighting that fully disordered proteins can be essential for cellular viability.

Maturation of protein-coding precursor messenger RNA (pre-mRNA) is closely linked to RNA polymerase II (Pol II) transcription. However, the mechanistic understanding of how pre-mRNA processing is coordinated with transcription remains incomplete. Conserved proteins interacting with the C-terminal domain of the largest catalytic subunit of Pol II and nascent RNA (CID-RRM factors) were demonstrated to play a role in pre-mRNA 3'-end processing and termination of Pol II transcription. Here, we use a fully reconstituted system to demonstrate that the fission yeast CID-RRM factor Seb1 acts as a bona fide elongation factor. Our analyses show that Seb1 exhibits context-dependent regulation of Pol II pausing, capable of either promoting or inhibiting pause site entry. We propose that CID-RRM factors coordinate Pol II transcription and pre-mRNA 3'-end processing by modulating the rate of Pol II transcription.

RNA performs many critical functions, nearly all of which are enabled by complex H-bonded structures. Nucleotides possess far fewer H-bond donors than acceptors, and the exocyclic amine is the only functional group that can donate two H-bonds, suggesting a specialized role. To assess the prevalence and structural contexts of dual-donating amines within structured RNAs, we created a computational workflow that mines and analyzes experimental RNA-containing structures. We evaluated H-bonding in over 250,000 amines from more than 1800 structures. Dual-donating amines were found most frequently in G's where they regularly interacted with diverse pairs of acceptors. In contrast, the dual-donating amines of A's and C's were less frequent, and they interacted with a more select set of acceptors. For all three nucleobases, amines that were dual-donating had both reduced solvent accessibility and higher atom density relative to amines that were single- or non-donating, indicating a tendency of dual donors to be more buried and help compact the RNA. Analysis of RNA pseudotorsion angles revealed that dual-donating amines are enriched in two non-A-form conformations, both of which are present in S-motifs found in the sarcin-ricin loop of rRNA. We find that dual-donating amines populate additional structural motifs including the GNRA tetraloop-receptor complex, the kink-turn, and the WC/H A-minor motif, which are present in the self-splicing group I intron, the SAM riboswitch, and the poly(A)-bound ENE. We suggest that dual-donating amines may enhance interactions by reducing conformational entropy loss of the RNA as well as strengthening nearby H-bonds.

Precise recognition of the boundaries between exons and introns (splice sites [SS]) is essential for the fidelity of gene expression. In contrast with the 5'SS, the consensus 3'SS sequence in both Saccharomyces cerevisiae and humans is just three nucleotides long: YAG. How the correct 3'SS is chosen among many possible alternates by the spliceosome is often unclear but likely involves proofreading by the Prp22 ATPase. In cryo-EM structures of spliceosome product (P) complexes, glutamine 1594 in the highly conserved α-finger domain of the Prp8 protein interacts directly with the -3 pyrimidine of the 3'SS. To investigate the role of this interaction, we constructed a Prp8^Q1594A mutant and studied the impact on splicing and 3'SS selection. Using splicing reporter assays and RNA-seq, we show that Prp8^Q1594A enables use of nonconsensus 3'SS by relaxing sequence requirements at the -3 and -2 positions. Consequently, this can change how adjacent 3'SS compete with one another during mRNA formation. The ability of Prp8^Q1594A to support splicing at non-YAG sites depends on the splicing factors Prp18 and Fyv6, and Prp8^Q1594A has genetic interactions with Prp22 mutants. Together, these findings suggest that the Prp8 α-finger acts as a sensor of 3'SS accommodation within the spliceosome active site. We propose that conformational change of the α-finger either allows or inhibits binding of the Prp22 C-terminal tail. This may provide a mechanism for regulating Prp22 activity in response to 3'SS binding.

Gene therapy using recombinant adeno-associated viral (AAV) vectors is a promising approach for treating inherited diseases. Precise characterization of AAV vector genomes and transcripts is essential for further optimization of this technique. Current visualization methods require multiple assays for detecting DNA and RNA, often involving mRNA-to-cDNA conversion. This can obscure insights into spatial distributions, particularly when AAV DNA and mRNA exhibit divergent trends. To address this challenge, we developed a padlock probe (PLP)-based rolling-circle amplification (RCA) technique. Using SplintR DNA ligase, which ligates single-stranded DNA splinted by complementary RNA sequences, enabled our method to directly target AAV mRNA without requiring conversion to cDNA, as well as genomic DNA. Incorporation of an intron within the transgene sequence allows probe designs that distinguish transgene DNA from its mRNA. This strategy enables specific detection of AAV single-stranded DNA (+), single-stranded DNA (-), and mRNA, each effectively amplified by PLP-RCA. Furthermore, this approach enables us to differentiate AAV single-stranded from double-stranded DNA by a combined treatment of lambda exonuclease and restriction enzyme digestion, providing the possibility of tracking the AAV genome processing following transduction. In transduced HeLa cells and liver tissues from AAV-injected mice, PLP-RCA revealed distinct temporal patterns of AAV DNA and mRNA localization, uncovering early DNA instability that influences transduction efficiency. This technique provides a robust and versatile platform for spatially resolved, single-cell analysis of AAV genome and transcript dynamics, facilitating a deeper understanding of AAV biology and aiding the optimization of vector-based gene therapies.

Small noncoding RNAs are versatile regulators of gene expression, capable of guiding the silencing of complementary mRNAs through their association with Argonaute proteins. This RNA-guided silencing, known as RNA interference (RNAi), is a conserved mechanism that shapes diverse biological processes, from development to genome defense. Central to the effectiveness of RNAi is the precise loading of small RNAs into their appropriate Argonaute partners, a step that ensures both specificity and fidelity in target recognition. Although most organisms harbor multiple classes of small RNAs and a corresponding repertoire of Argonautes, the rules that dictate their selective pairing remain only partially understood. Caenorhabditis elegans, with its expanded array of small RNA classes and Argonaute proteins, provides a powerful system to probe these mechanisms. In this review, we synthesize current knowledge on small RNA loading specificity, integrating insights from C. elegans with findings from other organisms. We focus on the interplay between small RNA biogenesis, biochemical properties of small RNAs, structural features of Argonautes, post-translational modifications, and the spatiotemporal co-expression patterns that together orchestrate precise Argonaute loading.

The transcription of human telomeres gives rise to a family of long noncoding RNAs, named TERRA. We previously showed that TERRA transcription is driven by CpG island promoters that are composed of stretches of three types of repeats. Using the human genome assembly that was available at that time, putative promoter sequences were localized at several subtelomeres. In this work, using the T2T-CHM13v2.0 human reference genome, we found that 39 out of 46 subtelomeres contain TERRA promoters and grouped them in classes depending on their organization. We then discovered 106 intrachromosomal TERRA-like promoters, adjacent to interstitial telomeric sequences (ITSs) or far away from them. Forty-seven of these promoters are flanked and may regulate the transcription of coding genes, ncRNAs or pseudogenes. Comparative sequence analysis showed that interstitial and subtelomeric promoters belong to a previously undescribed family of segmental duplications deriving from common ancestral sequences. RT-PCR experiments in seven cell lines demonstrated that TERRA transcripts can be synthesized from ITSs. TERRA expression was always low in primary fibroblasts and HeLa cells, while highly variable in the other two telomerase-positive (HT1080 and HEK293) and in the three telomerase-negative ALT cell lines (GM847, U2OS, and VA13). The analysis of RNA-seq data from U2OS, HeLa, and HEK293 cells showed that 205 ITSs were transcribed in at least one cell line. The fraction of transcribed ITSs and the level of their transcription increased with the length of the telomeric repeat stretch. Given the large number of transcribed ITSs, we propose that these loci contribute significantly to the production of the TERRA pool.

Alternative splicing (AS) enables cells to produce multiple protein isoforms from single genes, fine-tuning protein function across numerous cellular processes. However, despite its biological importance, researchers lack effective tools to compare the domain composition of AS-derived protein isoforms because such comparisons require both structural data and specialized methods. Recent advances in AI-driven protein structure prediction, particularly AlphaFold2, now make accurate structural determination of splicing isoforms accessible, enabling functional AS analysis at the protein structure level. Here, we present IsoformMapper, a web resource that analyzes AS through network community analysis of protein structures. This approach captures 3D physical interactions between protein regions often missed by traditional domain analysis, enabling structural comparisons of isoforms across any biological system. We illustrate our tool by analyzing validated human Bcl-X protein isoforms, revealing how AS creates distinct community structures with antagonistic functional roles. As a proof of concept, we apply our tool to investigate how GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling regulates plant de-etiolation through alternative splicing in Arabidopsis. In response to light, gun1 shows alterations in spliceosome component expression, suggesting that GUN1 contributes to AS regulation of genes essential for photosynthetic establishment. The gun1 mutant displays altered splice variant ratios for PNSL2, CHAOS, and SIG5. Our tool reveals that these isoforms form distinct protein community structures, demonstrating how AS impacts protein function and validating IsoformMapper's practical value.

Environmental stress activates transposons and is proposed to generate genetic diversity that facilitates adaptive evolution. piRNAs guide germline transposon silencing, but the impact of stress on the piRNA pathway is not well understood. In Drosophila, the Rhino-Deadlock-Cuff complex (RDC) drives transcription of clusters composed of nested transposon fragments, generating precursors that are processed into mature piRNAs in the cytoplasm. We show that acute heat shock triggers rapid, reversable loss of RDC localization and cluster transcript expression with coordinate changes in the cytoplasmic processing machinery. Maternal piRNAs bound to Piwi are proposed to guide Rhino localization to clusters during early embryogenesis. However, RDC re-localization after heat shock is accelerated in piwi mutants and delayed in thoc7 mutants, which disrupt piRNA precursor binding to THO complex, and we show that maternally deposited piRNAs are dispensable for RDC localization to the major 42AB cluster. Cluster specification is reconsidered in light of these findings.