RNA最新文献

Although protein-RNA interactions are crucial for many biological processes, predicting their binding free energies (ΔG) is a challenging task due to limited available experimental data and the complexity of these interactions. To address this issue, we developed a machine learning-based model designed to predict energy-based scores for protein-RNA complexes, called PANTHER score. By applying a local-to-global approach, the here proposed methodology can be subdivided into four steps: (1) we derived 87,117 pairwise local interaction energies out of 331,744 obtained from molecular dynamics simulations for a training set composed by 46 curated protein-RNA complexes; (2) we trained ML models derived from pairwise interaction features to predict the local interaction energies without performing MD runs; (3) we integrated the predicted local interaction energies with our here proposed local-to-global methodology, to calculate the model-specific PANTHER score; (4) we test the model-specific PANTHER score on a test set of 7 complexes (5) we further exposed all the models to an external stress set which includes 110 complexes with experimental ΔG allowing for final selection of the optimal model for implementation in the PANTHER scoring pipeline. Among all the multiple regression models developed here and evaluated on the test set, Random Forest Regression exhibited the highest predictive performance as a model-specific PANTHER score, with a Pearson correlation coefficient of (r) of 0.80 and mean absolute error (MAE) of 1.79 kcal/mol. Furthermore, the Random Forest Regression model maintained strong predictive capabilities on the stress set as well with (r) of 0.64 and MAE of 1.63 kcal/mol. Benchmarking against existing tools on the stress test set, the PANTHER score demonstrated superior accuracy and reliability. This study highlights the effectiveness of machine learning in addressing data limitations through innovative strategies, positioning here proposed PANTHER score as a valuable tool for predicting protein-RNA binding affinities in biomolecular research and drug discovery.

RNA 2'-phosphotransferase Tpt1 is a widely distributed enzyme that removes an internal RNA 2'-phosphate by transfer to NAD+. Tpt1 is essential in fungi, where it erases the 2'-PO4 mark installed by tRNA ligase during tRNA splicing. Tpt1 executes a two-step reaction in which: (i) the RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate and expel nicotinamide; and (ii) the ADP-ribose O2'' attacks the RNA 2'-phosphodiester to form 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate products. All Tpt1 enzymes studied to date are monofunctional units comprising a single bilobed fold composed of an RNA-binding lobe and an NAD+-binding lobe. We now find that fission yeast Tpt1 is an exception to this rule. Schizosaccharomyces pombe Tpt1 (SpTpt1) consists of an N-terminal RNA 2'-phosphotransferase catalytic domain (aa 1-237) linked to a C-terminal domain (aa 238-365) homologous to budding yeast iron-sulfur cluster assembly factor Yae1. The SpTpt1 catalytic domain and the Yae1 domain are both essential for S. pombe growth, though they need not be linked within the same polypeptide. A mutational analysis of the 2'-phosphotransferase domain illuminates the distinct contributions of essential active site constituents Arg50 and Arg96 during the two chemical steps of the Tpt1 pathway.

Eukaryotic translation initiation is critically regulated by 5' UTR features, including uORFs, Kozak sequences, and secondary structures, that modulate ribosome dynamics. Although canonical mRNAs dominate protein synthesis, ribosome profiling and peptidomics reveal ribosomes actively engaging putative non-coding RNAs (ncRNAs), translating enigmatic short ORFs (sORFs). We systematically analyzed 5' UTR architectures across canonical mRNAs, ribosome-associated ncRNAs (translationally active), and non-translated ncRNAs using curated human datasets. mRNAs exhibited optimal translational features (short 5' UTRs, few uORFs), while translated ncRNAs showed intermediate features, and non-translated ncRNAs the weakest. Notably, mRNAs with long 5' UTRs maintained high translational efficiency through conserved regulatory elements. Integrating these features into our newly developed random forest model, plusCE, surpassed existing methods in predicting translation efficiency, suggesting their potential relevance to translation mechanisms and providing guidance for rational 5' UTR design to modulate translation. Although some ncRNAs are frequently bound by ribosomes, they show no evidence of stable translation, consistent with their lack of coding-related evolutionary signatures. Our analysis suggests that ribosome-bound ncRNAs may not reflect adaptive evolution toward coding function, but rather represent a reservoir of untranslated transcripts that engage the translation machinery through permissive sequence features. Together, these results demonstrate that ribosome engagement is primarily shaped by 5' UTR sequence features, highlighting the importance of regulatory grammar in translation control and complementing current models of ncRNA evolution.

Small RNAs play essential roles in gene regulation across diverse biological processes. Crosslinking, ligation, and sequencing of hybrids (CLASH) experiments have revealed that PIWI and Argonaute proteins can each bind a wide range of mRNA targets with distinct base-pairing rules, raising questions about the flexibility and functional relevance of these interactions. Given that crosslinking-induced mutations (CIMs) provide single-nucleotide resolution molecular footprints of RNA-binding proteins, we developed MUTACLASH, a bioinformatics tool for systematically analyzing CIMs in CLASH datasets. Our analyses indicate that CIMs function as molecular footprints of Argonaute binding on target mRNAs. Specifically, for C. elegans miRNA and piRNA CLASH data, CIMs are enriched at the center of small RNA binding sites, as well as at nucleotides within mRNA target sites that exhibit local mismatches in piRNA interactions. Furthermore, we show that mRNAs with non-canonical miRNA and piRNA binding sites and/or low hybrid abundance marked by CIMs exhibit stronger regulatory effects than those without CIMs, demonstrating the utility of CIM analysis in identifying functional small RNA binding sites, including those that are otherwise likely overlooked with current analysis tools.

In the twenty years since the first genome sequencing of Aspergillus fumigatus, the field has seen an explosion in both the number of sequenced genomes and our molecular understanding of this ubiquitous human fungal pathogen. Despite an improved knowledge of the A. fumigatus genome, we still know little about the transcriptome, with key regulatory sequences like the untranslated regions of mRNA based only on in silico predictions and bulk-RNA-seq. Here, we provide an improved description of 5' and 3' untranslated regions of A. fumigatus poly(A)-enriched RNA through experimental mapping of transcription start sites and polyadenylation sites using 5' and 3' End-Seq. We assigned high-quality 5' ends to 2,747 genes (average length 126 nt), 3' ends to 7,079 genes (average length 268 nt), and improved our understanding of the regulatory landscape of A. fumigatus gene expression. We leveraged the refined 5' UTRs to identify upstream open reading frames and binding sites for important RNA binding proteins like the translational regulator Ssd1 and the 3' UTRs to define binding sites for PUF proteins known to contribute to mRNA localization and regulation. Although a single isoform typically dominated expression, we observed 148 instances of alternative start sites and 1,675 alternative stop sites. Interestingly, we detected multiple examples of premature transcriptional termination, including the first evidence for promoter-proximal premature transcriptional termination in a member of the Eurotiomycetes. Ultimately, we provide a resource to the Aspergillus community and an accurate starting point for unravelling the complexities of gene regulation in an important human pathogen.

Hepatitis C virus (HCV) is a major global health burden, associated with chronic liver diseases including cirrhosis and hepatocellular carcinoma. Viral replication critically depends on conserved cis-acting RNA elements (CREs), such as the 5BSL3.2 stem-loop near the 3' end of the open reading frame. This element forms a long-range kissing-loop interaction with the SL2 domain of the 3'X tail, essential for efficient genome replication. However, the role of host RNA-binding proteins (RBPs) in regulating this RNA-RNA interaction remains poorly understood. To explore this, we investigated whether the host RBP hnRNPA1 modulates HCV replication by targeting the 5BSL3.2 element. Using an integrated approach combining structural biology, biophysics, and biochemical assays, we identify the terminal loop of 5BSL3.2 as a high-affinity binding site for the tandem RNA recognition motifs (RRMs) of hnRNPA1. Our data reveal that adenine-rich residues within the loop are critical for binding specificity. Our results uncover a structural mechanism by which hnRNPA1 binding perturbs the kissing-loop interaction between 5BSL3.2 and the SL2 element of the viral 3'X-tail, which impacts viral replication. This study highlights a previously unrecognized role of hnRNPA1 in modulating viral RNA structure and suggests a novel interface for host-directed antiviral intervention.

Magnesium is vital for bacterial survival, and its homeostasis is tightly regulated. Intracellular pathogens like Mycobacterium tuberculosis (Mtb) often face host-mediated magnesium limitation, which can be counteracted by upregulating the expression of Mg²⁺ transporters. This upregulation may be via Mg²⁺-sensing regulatory RNA such as the Bacillus subtilis ykoK Mbox riboswitch, which acts as a transcriptional "OFF-switch" under high Mg²⁺ conditions. Mtb encodes two Mbox elements with strong similarity to the ykoK Mbox. In the current study, we characterize the Mbox encoded upstream of the Mtb pe20 operon, which is required for growth in low Mg²⁺/low pH. We show that this switch operates via a translational expression platform and Rho-dependent transcription termination, which is the first such case reported for an Mbox. Moreover, we show that the switch directly controls a small ORF encoded upstream of pe20 We have annotated this highly conserved uORF rv1805A, but its role remains unclear. Interestingly, a homologous gene exists outside the Mbox-regulated context, suggesting functional importance beyond magnesium stress. Overall, this study uncovers a dual mechanism of riboswitch-regulation in Mtb, combining translational control with Rho-mediated transcription termination. These findings expand our understanding of RNA-based gene regulation in mycobacteria, with implications for pathogenesis and stress adaptation.

The 5'-N7-methylated guanosine triphosphate cap structure plays a critical role in mRNA translation and mRNA stability. The recent invention of co-transcriptional capping of mRNAs using Trinucleotide Capped Primers (TCP) allowed for development of large-scale in vitro transcription (IVT) synthesis of mRNA carrying a eukaryotic Cap 1 structure (TCP-mRNA). Here we present a novel "one-pot-two-step" methodology for the synthesis of TCPs that improves the yield and simplifies the isolation and purification of the TCPs. Over 70 different modified TCPs, the analogs of 7mGpppAmpG trimer, were synthesized, characterized, and tested for their ability to initiate IVT reaction. Results demonstrate that full complementarity of TCP to a template strand of dsDNA template at transcription initiation (start) site, at positions +1 and +2, is required and sufficient to obtain capped TCP-mRNA with high capping efficiency (>98%) and high yield (>5 mg/mL). The developed approach can be applied for small- and large-scale mRNA synthesis carrying various 5'-cap structures.

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous enzyme cofactor that serves as a carrier of hydride ions for metabolic oxidation-reduction reactions. NAD is also sometimes used as a source of activated adenosine monophosphate (AMP) for adenylation reactions or as a precursor of ADP-ribose upon removal of nicotinamide. Many bacterial riboswitch classes are known to sense nucleotide-derived enzyme cofactors, but NAD is one of several ancient cofactors that have few or no known riboswitch representatives. Two rare riboswitch classes, named NAD+-I and NAD+-II, have been reported that regulate genes relevant to NAD biosynthesis and transport. However, these RNAs exhibit unusual functional and structural properties. Here we report that miniature NAD+-II riboswitches, named mini-NAD+-II, are more abundant and widespread than the longer RNAs that were used to defined the original consensus model for this class. The newfound examples are commonly found within lactic acid bacteria, which are notable for varied metabolic fermentation strategies used to maintain sufficient NAD+. Furthermore, the simple H-type pseudoknot core of mini-NAD+-II aptamers is similar to that of class I preQ1 riboswitch (preQ1-I) aptamers. Thus, H-type pseudoknots might serve as a versatile architecture for the natural or synthetic construction of ligand-binding aptamers.

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.