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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.

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, we proposed a methodology further subdivided into five steps: (1) We derived 87,117 pairwise local interaction energies from 331,744 MD-derived interactions across 46 curated protein-RNA complexes; (2) we trained ML models on pairwise interaction features to predict local interaction energies without performing MD simulations; (3) we integrated predicted local interaction energies using a local-to-global methodology, to compute model-specific PANTHER Score; (4) we evaluate model-specific PANTHER Score on an independent test set of seven complexes; and (5) we validated and selected the optimal model using an external stress set of 110 complexes with experimental ΔG values for implementation in the PANTHER Scoring pipeline. Among the regression models developed, Random Forest Regression exhibited the highest predictive performance as a model-specific PANTHER Score, achieveing a Pearson correlation (r) of 0.80 and MAE of 1.79 kcal/mol on the test set. It maintained strong predictive capabilities on the stress set (r = 0.64, MAE = 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 MD and machine learning in addressing data limitations through innovative strategies, positioning the PANTHER Score as a robust tool for predicting protein-RNA binding affinities in biomolecular research, drug discovery and mainly in RNA-therapeutics.

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 replication 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.

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.

The 5'-N ⁷-methylated guanosine triphosphate cap structure plays a critical role in mRNA translation and mRNA stability. The recent invention of cotranscriptional capping of mRNAs using trinucleotide capped primers (TCPs) allows 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 a ^7mGpppA_mpG trimer, were synthesized, characterized, and tested for their ability to initiate IVT reaction. The 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). This approach can be applied from small- to large-scale mRNA synthesis carrying various 5'-cap structures.

Numerous RNA modifications are known in prokaryotes, but their dynamics and function in regulation remain largely unexplored. In Escherichia coli, three methyltransferases catalyze the 5-methylcytosine (m⁵C) modification in ribosomal RNA. Here, we introduce m⁵C-rolling circle loop-mediated isothermal amplification (m⁵C-Rol-LAMP) as a novel qPCR-based method that offers high sensitivity and site-specific resolution to detect and quantify m⁵C in total RNA. When applying m⁵C-Rol-LAMP to E. coli under heat stress (45°C), we observe a site-specific increase of m⁵C at position 1407 of 16S rRNA from 77% to 89%, while m⁵C levels at positions 967 (16S) and 1962 (23S) remain unchanged. In recovered cells (at 37°C), the m⁵C abundance partially returns to the no stress level. Under oxidative stress, the level of m⁵C1407 also increases, but remains high in recovered cells. These results demonstrate for the first time a reversible, stress-dependent and site-specific change in the rRNA modification level of a bacterium. m⁵C-Rol-LAMP is a powerful and easy-to-use tool for studying m⁵C in all RNA species, allowing the quantitative and site-specific detection of this modification.

Eukaryotic translation initiation is critically regulated by 5' UTR features, including uORFs, Kozak sequences, and secondary structures, which modulate ribosome dynamics. Although canonical mRNAs dominate protein synthesis, ribosome profiling and peptidomics reveal ribosomes actively engaging putative noncoding RNAs (ncRNAs), translating enigmatic short ORFs (sORFs). We systematically analyzed 5' UTR architectures across canonical mRNAs, ribosome-associated ncRNAs, and nontranslated ncRNAs using curated human data sets. mRNAs exhibited optimal translational features (short 5' UTRs, few uORFs), while ncRNAs with translation-associated signals showed intermediate features, and nontranslated 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 data sets. Our analyses indicate that CIMs function as molecular footprints of Argonaute binding on target mRNAs. Specifically, for Caenorhabditis 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 noncanonical 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 20 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 2747 genes (average length 126 nt), 3' ends to 7079 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 1675 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 unraveling the complexities of gene regulation in an important human pathogen.

Epithelial cells exhibit a highly polarized organization along their apico-basal axis, a feature that is critical to their function and is frequently perturbed in cancer. One less explored process modulating epithelial cell polarity is the subcellular localization of mRNA molecules. In this study, we report that several mRNAs encoding evolutionarily conserved epithelial polarity regulatory proteins, including Zo-1, Afdn and Scrib, are localized to cell junction regions in Drosophila epithelial tissues and human epithelial cells. Targeting of these mRNAs coincides with robust junctional distribution of their encoded proteins, and these transcripts are translated in proximity to cell junction regions. Through systematic immuno-labeling, we identify a collection of RNA binding proteins with cell junction distribution patterns, several of which associate with junctional transcripts and are functionally required for proper targeting of ZO-1 and SCRIB proteins. Loss-of-function of two candidate factors, MAGOH and PCBP3, differentially impacts junctional mRNA, with MAGOH knock-down reducing Zo-1 and Scrib transcript targeting and localized translation, while PCBP3 knock-down only perturbs local translation. Depletion of Drosophila MAGO in vivo in follicular epithelial cells also disrupts the distribution of junctional transcripts and proteins. Finally, through tissue microarray analysis of ovarian cancer tumor specimens, we find that the expression of MAGOH and ZO-1 is positively correlated and that both proteins are potential biomarkers of good prognosis. We conclude that localized mRNA regulation at cell junction regions is important for modulating epithelial cell integrity.