RNA最新文献

RNA-binding proteins (RBPs) are composed of RNA-binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analyses have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in vivo binding pattern is poorly explored. Here, we upscaled the RNA-tagging method to map the transcriptome-wide binding of 16 RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding. However, multiple IDRs that lacked predicted RNA-binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in vivo.

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO₄ and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO₄ and 5'-PO₄ termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO₄ and 5'-PO₄ termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg²⁺ that illuminates the basis for guanosine nucleotide specificity.

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN-binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process.

Eukaryotic genomes typically encode one member of the DXO/Dxo1/Rai1 family of enzymes, which can hydrolyze the 5' ends of RNAs with a variety of structures that deviate from the canonical ^7mGpppN. In contrast, the Saccharomyces genome encodes two family members and the second copy, Dxo1, is a distributive 5' exoribonuclease that is required for the final maturation of the 5' end of 25S rRNA from a 25S' precursor. Here we show that this 25S rRNA maturation function is not conserved across kingdoms, but arose in the budding yeasts. Interestingly, the origin of 25S processing capacity coincides with the duplication of this gene, and this capacity is absent in the nonduplicated genes. Strikingly, two different clades of budding yeasts have undergone parallel evolution: Both duplicated their DXO/Dxo1/Rai1 gene, and in both cases, one copy gained the 25S processing function. This was accompanied by many parallel sequence changes, a remarkable case of reproducible neofunctionalization.

Folded RNAs contain tertiary contact motifs whose structures and energetics are conserved across different RNAs. The transferable properties of RNA motifs simplify the RNA folding problem, but measuring energetic and conformational properties of many motifs remains a challenge. Here, we use a high-throughput thermodynamic approach to investigate how sequence changes alter the binding properties of naturally occurring motifs, the GAAA tetraloop • tetraloop receptor (TLR) interactions. We measured the binding energies and conformational preferences of TLR sequences that span mutational pathways from the canonical 11ntR to two other natural TLRs, the IC3R and Vc2R. While the IC3R and Vc2R share highly similar energetic and conformational properties, the landscapes that map the sequence changes for their conversion from the 11ntR to changes in these properties differ dramatically. Differences in the energetic landscapes stem from the mutations needed to convert the 11ntR to the IC3R and Vc2R rather than a difference in the intrinsic energetic architectures of these TLRs. The conformational landscapes feature several nonnative TLR variants with conformational preferences that differ from both the initial and final TLRs; these species represent potential branching points along the multidimensional sequence space to sequences with greater fitness in other RNA contexts with alternative conformational preferences. Our high-throughput, quantitative approach reveals the complex nature of sequence-fitness landscapes and leads to models for their molecular origins. Systematic and quantitative molecular approaches provide critical insights into understanding the evolution of natural RNAs as they traverse complex landscapes in response to selective pressures.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Despite its severe phenotypes, studies in mouse models suggest that restoring MeCP2 levels can reverse RTT symptomology. Nevertheless, traditional gene therapy approaches are hindered by MeCP2's narrow therapeutic window, complicating the safe delivery of viral constructs without overshooting the threshold for toxicity. The 3' untranslated region (3' UTR) plays a key role in gene regulation, where factors like miRNAs bind to pre-mRNA and fine-tune expression. Given that each miRNA's contribution is modest, blocking miRNA binding may represent a potential therapeutic strategy for diseases with high dosage sensitivity, like RTT. Here, we present a series of site-blocking antisense oligonucleotides (sbASOs) designed to outcompete repressive miRNA binding at the MECP2 3' UTR. This strategy aims to increase MeCP2 levels in patients with missense or late-truncating mutations, where the hypomorphic nature of the protein can be offset by enhanced abundance. Our results demonstrate that sbASOs can elevate MeCP2 levels in a dose-dependent manner in SH-SY5Y and patient fibroblast cell lines, plateauing at levels projected to be safe. Confirming in vivo functionality, sbASO administration in wild-type mice led to significant Mecp2 upregulation and the emergence of phenotypes associated with Mecp2 overexpression. In a T158M neural stem cell model of RTT, sbASO treatment significantly increased MeCP2 expression and levels of the downstream effector protein brain-derived neurotrophic factor (BDNF). These findings highlight the potential of sbASO-based therapies for MeCP2-related disorders and advocate for their continued development.

Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm⁵), 5-methoxycarbonylmethyl (mcm⁵), or 5-methoxycarbonylhydroxymethyl (mchm⁵) side-chain. The presence of these side-chains allows proper pairing with cognate codons, and they are particularly important in tRNA species where the U₃₄ residue is also modified with a 2-thio (s²) group. The first step in the synthesis of the ncm⁵, mcm⁵, and mchm⁵ side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1, which encodes the cytosolic Cu, Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-modified U₃₄ residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNA^Phe Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells.

Prominent members of the Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation by dimethylating two nucleotides of small subunit rRNA including the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.

Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.