RNA最新文献

Ribosome profiling (Ribo-seq) is a next-generation, high-resolution sequencing technique that captures ribosome-protected mRNA fragments to map ribosome positions across the transcriptome. This method serves as a powerful proxy for global translational activity by revealing where ribosomes engage with mRNAs. Recent advances have expanded the utility of Ribo-seq to resolve distinct ribosome populations, including initiating ribosomes, small subunits, collided ribosomes, mitochondrial ribosomes, and those associated with specific translation factors or localized to subcellular compartments. These methodological advances have significantly broadened the scope of Ribo-seq, enabling new insights into the molecular mechanisms that govern translation across diverse eukaryotic systems. In this mini-review, we highlight key innovations in Ribo-seq technology and discuss how they have deepened our understanding of the spatial, temporal, and regulatory dimensions of translational control.

The piRNA biogenesis machinery localizes to phase separated nuage granules, but nuage function is not well understood. We therefore assayed nuage composition, piRNA expression and transposon silencing in Drosophila mutants that disrupt piRNA precursor production and nuclear export, ping-pong amplification and phased piRNA biogenesis. These mutations destabilize the genome and activate Chk2 signaling and chk2/mnk double mutants were therefore analyzed in parallel. Aub and Vasa are required for ping-pong amplification and Armi promotes phased piRNA processing. We show that Chk2 activation releases Aub and Vasa from nuage and that piRNA precursors are required for nuage localization of the ping-pong and phased biogenesis machinery. However, this analysis also indicates that Vasa, Aub, and Armi concentration in nuage is dispensable for piRNA production and transposon silencing, indicating that dispersed cytoplasmic proteins can drive these processes. We speculate that nuage sequesters silencing effectors, which are released by Chk2 in response to transposon mobilization.

In most eukaryotes, sense/antisense RNA duplexes can be processed into small interfering RNAs by the ribonuclease III Dicer, a key component of the RNA interference (RNAi) machinery, which has been lost by the budding yeast Saccharomyces cerevisiae Previous studies in this species revealed the pervasive formation of double-stranded (ds) RNA involving antisense Xrn1-sensitive long noncoding (lnc) RNAs, which interferes with their degradation through translation-dependent nonsense-mediated mRNA decay (NMD). However, apart from S. cerevisiae, little is known about the post-transcriptional metabolism of lncRNAs, in particular the functional impact of RNAi. Herein, we profiled NMD targets in Naumovozyma castellii, a budding yeast endowed with cytoplasmic RNAi. We identified 592 lncRNAs accumulating in a mutant of the NMD core factor Upf1. Most of them also accumulate in other NMD mutants and upon translation elongation inhibition, indicating a translation-dependent degradation mechanism. Consistently, Ribo-seq analyses confirmed ribosomes binding for a fraction of them. Within the coding transcriptome, we found that the Dicer-coding mRNA is also regulated by NMD. The resulting upregulation of DCR1 in NMD-deficient cells correlates with an increased production of small RNAs from dsRNA-forming NMD-sensitive lncRNAs and mRNAs. Finally, we observed that Dicer inactivation in Upf1-lacking cells attenuates the accumulation of dsRNA-forming NMD targets. Together, our data highlight the conserved roles of NMD and translation in the post-transcriptional metabolism of lncRNAs and provide insight into the functional impact of endogenous RNAi on the transcriptome.

The RNase II/RNB family of exoribonucleases is present in all domains of life and includes three main eukaryotic members, the Dis3-like proteins (Dis3, Dis3L1, and Dis3L2). At the cellular level, Dis3L2 is distinguished by the unique preference for uridylated RNA substrates and the highest efficiency in degrading double-stranded RNA. Defects in these enzymes have been linked to some types of cancers and overgrowth disorders in humans. In this work, we used the Dis3L2 protein from the model organism Schizosaccharomyces pombe (SpDis3L2) to better understand the mechanism of action of Dis3-like exoribonucleases, and to elucidate how single amino acid substitutions in these proteins can affect the biochemical properties of the enzymes, potentially contributing to the molecular basis of the related human diseases. We determined the crystal structure of SpDis3L2 bound to a U₁₃ RNA, in which the protein displays a typical vase-like conformation, accommodating 6 nucleotides of the RNA 3'-end. Furthermore, we constructed two SpDis3L2 protein variants, harboring single amino acid substitutions mimicking the ones already found in human patients, to test their catalytic activity in vitro. We highlight the A756R SpDis3L2 variant, which loses the ability to degrade double-stranded RNA substrates and accumulates intermediate degradation products when degrading single-stranded RNA substrates. As such, A756 seems to be a key residue responsible for the normal cellular function of Dis3L2, specifically regarding its important role in the degradation of structured RNA substrates.

Riboswitches are noncoding mRNA regions that regulate gene expression by sensing small molecules. While most riboswitches turn off gene expression, guanidine-I family riboswitches enhance gene expression. Although crystal structures provide insights into the structural basis for guanidinium ion (Gdm⁺) sensing by the guanidine-I riboswitch aptamer (GRA) domain, the mechanistic interplay between ligand and metal ion binding, RNA conformational changes, and regulatory function remains poorly understood. Using molecular dynamics simulations, we explore the combined effects of the positively charged Gdm⁺, Mg²⁺, and K⁺ observed in close proximity in the experimental crystal structure on the GRA structural dynamics. Our simulations reveal that the binding pocket frequently transitions between ligand bound-like and unbound-like states in the absence of divalent ions, while Mg²⁺ stabilizes a bound-like RNA conformation. Furthermore, both Mg²⁺ and Gdm⁺ facilitate K⁺ positioning near the binding pocket. As a result, Mg²⁺, Gdm⁺, and K⁺ synergistically increase the structural rigidity of the GRA domain, particularly the P2-P3 junction and the 3' end near the terminator stem. This enhances localized interactions that pull the P1a and P3 domains together to make the transcriptional control region available for expression. Our proposed mechanism is fully consistent with experimental structural and biochemical (including isothermal titration calorimetry and structure-guided mutagenesis) data and rationalizes how the unique triad of ions works together to influence the conformational dynamics of the aptamer domain and riboswitch function. This information can guide future synthetic riboswitch design and the identification of novel therapeutic targets beyond static structural information.

Noncoding introns are a unifying feature of protein-coding genes in virtually all extant eukaryotes, with most lineages following the canonical intron structure. However, euglenozoans, unicellular flagellates that include free-living euglenids, human pathogenic kinetoplastids, and highly diverse and abundant marine diplonemids, are a notable exception. Euglenozoan genomes range from extremely intron-poor kinetoplastids to euglenid genomes containing both canonical and noncanonical introns. Here, we present a comprehensive analysis of splice sites and spliceosomal components in six species of understudied diplonemids. All diplonemids examined contain a nearly complete set of spliceosomal snRNP components, indicating the presence of a functional U2-type spliceosome. However, the majority of introns in the hemistasiid diplonemids Artemidia motanka and Namystynia karyoxenos are noncanonical and lack conserved GT-AG terminal dinucleotides typical for U2-type introns. These noncanonical introns are capable of extensive base-pairing, which brings intron ends into close proximity. Thus, while the splicing apparatus is conserved in diplonemids, the splice sites are highly variable among individual species.

The spliceosome is a highly dynamic structure that undergoes continuous structural alterations through the sequential association and dissociation of small nuclear RNAs and protein factors during precursor mRNA splicing. These structural changes are driven by eight DExD/H-box RNA helicases that act at distinct stages of the splicing cycle. Among them, Prp5 and Sub2 are involved in prespliceosome formation, with Prp5 implicated in displacing the U2 snRNP component Cus2, and Sub2 in facilitating the release of the Msl5-Mud2 heterodimer. However, the precise mechanisms underlying the functions of these two proteins remain unclear. Here, we show that Sub2 is not essential for splicing in vitro, but it can enhance splicing independently of ATP. Strikingly, prespliceosome formation can proceed without ATP in the absence of either Sub2 or Cus2. These findings reveal a coordinated interplay among Prp5, Sub2, Cus2 Mud2, and Msl5 during prespliceosome formation.

T-box riboswitches belong to a specific class of RNA regulatory elements that control gene expression in Gram-positive bacteria, including prominent human pathogens. They sense the availability of amino acids by detecting the aminoacylation status of their cognate tRNAs and regulate the expression of genes involved in aminoacylation, amino acid transport, and metabolism. Recent advances in the structures and mechanisms of several regulatory noncoding RNAs among pathogenic bacteria have garnered attention for the development of a new generation of species-specific antibacterials. The frequently acquired resistance against current antibiotics has emerged as a significant challenge for healthcare systems and a serious threat to public health. Herein, we report the characterization of an effective T-box riboswitch inhibitor, termed T-box-i, which efficiently disrupts T-box riboswitch-mediated transcription in vivo. T-box-i was selected through a virtual screening campaign of commercially available small molecules against high-resolution crystallographic structures of T-box riboswitches. It exhibited no cytotoxicity in mammalian cells nor induced antibiotic resistance in Staphylococcus aureus cultures. These findings provide valuable insights into exploiting T-box riboswitches as antibiotic targets and underscore the therapeutic potential of compounds that selectively target extensively structured regulatory RNA elements and interfaces to combat drug-resistant pathogens.

Trl1-type ligases play an essential role in fungi and plants during the nonconventional tRNA splicing as well as the unfolded protein response. The tripartite enzyme consists of an N-terminal adenylyltransferase domain (LIG), a central polynucleotide kinase domain (KIN), and a C-terminal cyclic phosphodiesterase domain (CPD). The Trl1-mediated reaction can be divided into two steps: (1) RNA end modification by the KIN and CPD domains, and (2) the adenylyltransferase reaction catalyzed by the LIG domain resulting in the phosphodiester bond formation. Due to its absence in humans, Trl1 is often discussed as a potential target for antifungal therapy. To date, structural information on the full-length Trl1 is missing. Several crystal structures of the individual LIG and KIN as well as a KIN-CPD construct have been solved, thereby elucidating the fold of the individual domains, their cofactor, and substrate binding. Here, we provide the missing crystal structure of the two-domain LIG-KIN construct from the thermophilic fungus Chaetomium thermophilum, revealing the interdomain assembly and interface. Based on our structure and complementing AlphaFold3 predictions, we further propose a model with implications for interdomain RNA substrate transfer.

In addition to their function in protein synthesis, translating ribosomes serve as sensors that communicate the presence of aberrant messenger RNAs (mRNAs); however, how they recognize damage to their ribosomal RNA (rRNA) remains poorly understood. The conserved sarcin/ricin loop (SRL) of the 25S rRNA is a component of the GTPase center essential for ribosome movement during translation. In this study, we expressed an RNA N-glycosylase called pokeweed antiviral protein (PAP) in yeast Saccharomyces cerevisiae to specifically damage rRNA by hydrolysis of a purine base from the SRL. 25S rRNA depurination inhibited translation elongation, as shown by reduced incorporation of a methionine analog and binding of eukaryotic elongation factor 2 (eEF2) to ribosomes. PAP expression altered sucrose gradient profiles, increasing free subunits and 80S peaks and reducing polysomes without causing ribosome collisions. We discovered depurinated rRNA associated with 80S monosomes and polysomes, suggesting that cells would detect damage to rRNA during active translation. These ribosomes were ubiquitinated by E3 ligases Mag2 and Hel2, elements of the 18S nonfunctional rRNA decay (NRD) pathway involved in recognizing slow-moving ribosomes. Furthermore, mass spectrometry analysis revealed ubiquitination of ribosomal protein uS3, characteristic of 18S NRD. Even though the SRL is a component of the large ribosomal subunit, its depurination is signaled by ubiquitin ligases that recognize damage to the small subunit. We suggest that slow translation elongation is the factor that communicates SRL depurination to E3 ubiquitin ligases, which extends our understanding of how rRNA integrity is surveilled in yeast.