RNA最新文献

Argonaute proteins mediate gene silencing via small regulatory RNAs that are generated by distinctive biogenesis pathways. In animals, three main classes are recognized: ~21-24 nucleotide (nt) microRNAs (miRNAs), ~21-24 nt small-interfering RNAs (siRNAs) and ~24-32 nt Piwi-interacting RNAs (piRNAs). Mechanistic understanding of these pathways was gained from genetic, biochemical and genomic studies in a handful of model systems, where key ribonucleolytic events were identified that specify stereotyped positioning of small RNAs relative to their precursor transcripts. With burgeoning availability of assembled genomes and small RNA data, there are abundant opportunities to characterize the diversity of small RNAs across non-model organisms. While several tools are well-suited to analyze specific small RNA pathways, an integrated package that can help classify and interpret all three major classes of small RNAs is wanting. To address this need, we developed a simple and efficient R package (MiSiPi.Rna) that can generate a variety of plots and statistics for pre-selected loci, which enable the characterization of diverse biogenesis features of miRNAs, siRNAs and piRNAs. MiSiPi.Rna requires minimal computational expertise to run, and will facilitate efforts to annotate and analyze the major classes of Argonaute-based small regulatory RNAs in arbitrary species of choice.

Many non-protein-coding RNAs have been discovered and more are being discovered each year. At first we know them only by their sequences in a few organisms, but to understand their function and interactions, we need to understand what 3D structures they may form, in whole or in part. Many hairpin and internal loops are known to form structured 3D motifs, many of which are recurrent across different non-coding RNAs, for example, kink turn and sarcin-ricin internal loops, and GNRA, UNCG, and T-loop hairpin loops. As such, a new non-protein-coding RNA may well have one or more known structured 3D loop motifs. The goal of this paper is to introduce a tool which can identify loops in Rfam seed alignments that match well to known 3D loop motifs, and which makes those identifications easily accessible. JAR3D was developed to map sequences of hairpin and internal loops to known 3D motifs, and was extended for this work to 3-way and 4-way junction motifs. We applied JAR3D to 4,166 Rfam seed alignments from Rfam 15.0 and made the results accessible on the JAR3D web page, which makes it easy to inspect and evaluate the possible matches for each loop in each Rfam family. We provide several examples which validate JAR3D's ability to identify the correct loop motif, using 3D structures of RNAs outside of the training set. We created a new page to search for instances of a particular loop motif across all Rfam families, to facilitate studies of how widespread the occurrence of each motif is. We provide statistics on how many Rfam loops appear to match well to a known 3D motif. Match rates are much higher for internal loops than for hairpins or multi-helix junctions.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, primarily causes diarrhea in piglets and has the potential for cross-species transmission to humans. The recent detection of PDCoV in Haitian children underscores the urgent need for developing antiviral therapeutic strategies. G-quadruplexes (G4s) are implicated in the modulation of viral infection; however, their identification and roles in the PDCoV life cycle remain unclear. Here, we identified a highly conserved G4 structure, designated PDCoV-G4, located within the coding region of PDCoV non-structural protein 8 (nsp8). PDS and TMPyP4, two well-known G4-binding ligands, were found to target PDCoV-G4 and exhibit anti-PDCoV activity. Interestingly, PDS stabilizes the structure of PDCoV-G4, while TMPyP4 disrupts it. The recombinant PDCoV with G4-disruptive mutations (rPDCoV-nsp8mut) displays resistance to both PDS and TMPyP4. Utilizing an embryonated chicken eggs (ECEs) infection model, we observed that TMPyP4 provides superior protective effects for rPDCoV-wt-infected ECEs compared to PDS. However, both PDS and TMPyP4 exhibited diminished protective effects on chicken embryos infected with rPDCoV-nsp8mut, relative to rPDCoV-wt, further confirming their in vivo antiviral activity through targeting PDCoV-G4. These findings demonstrate that the PDCoV-G4 plays a crucial regulatory role in the PDCoV life cycle and pathogenicity, representing a potential target for antiviral therapy.

Membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Many MLOs remain spatially compartmentalized, avoiding fusion or engulfment. MLOs are formed by dynamic multivalent interactions, often mediated by proteins with intrinsically disordered regions (IDRs). However, the molecular principles behind how IDRs maintain MLO independence remain poorly understood. Here, we investigated the proline/glutamine (P/Q)-rich IDR of SFPQ, a protein identified as a key factor in segregating paraspeckles from nuclear speckles. Paraspeckle segregation analyses, using SFPQ mutants tethered to NEAT1_2 long noncoding RNA, revealed that P/Q residues within the SFPQ IDR, conserved from humans to zebrafish, are crucial for its segregation activity. Beyond amino acid composition, the blocky patterns of P/Q residues, in which proline- and glutamine-rich blocks are repetitively arranged, are required for the segregation from nuclear speckles. Among human IDRs exhibiting PQ-block patterns, BRD4 IDR shows strong sequence similarity to the SFPQ IDR, and exhibits comparable segregation activity. Molecular dynamics simulation suggests that the PQ-blocky patterns required for the paraspeckle segregation do not correlate with the IDR characteristics necessary for self-assembly. Thus, these data suggest that the PQ-blocky patterns in IDRs represent a previously uncharacterized property that contributes to MLO independence, possibly through a mechanism distinct from the conventional phase separation-promoting function of IDRs.

mRNA-based therapeutics are commonly produced through T7 RNA Polymerase-mediated in vitro transcription. Introducing these exogenous RNAs into human cells activates an RNA sensor Protein Kinase R (PKR), which suppresses translation initiation and reduces their therapeutic effectiveness. Incorporating uridine analogs into these transcripts prevents PKR activation and translation shutdown, but the underlying mechanism remains unclear. Here, we demonstrate that treating T7 RNA Polymerase-produced transcripts with RNase III, which selectively degrades double-stranded RNA (dsRNA), blocks PKR activation and downstream translation-inhibition events, including eIF2α phosphorylation and stress granule formation in human cells. Interestingly, dsRNAs generated with uridine analogs robustly induce eIF2α phosphorylation and stress granules to the same extent as dsRNA containing uridine. These findings indicate that uridine analogs do not prevent PKR from detecting dsRNA. Instead, we show that uridine analogs decrease the production of T7 RNA Polymerase byproducts, including antisense RNA and dsRNA, which activate PKR and downstream stress responses. Finally, we demonstrate that higher amounts of exogenous RNA, lacking T7 RNA Polymerase byproducts, can induce stress granules independently of PKR and phospho-eIF2α, but dependent on stress granule scaffold proteins G3BP1 and G3BP2. Together, our findings show that uridine analogs mitigate PKR signaling not by blocking mRNA-PKR interactions, but by minimizing dsRNA byproducts from T7 Polymerase transcription. Furthermore, stress granule formation in response to high levels of exogenous RNA can occur through a mechanism that does not depend on PKR but relies on G3BP1 and G3BP2. These insights clarify the role of uridine analogs in PKR activation and may inform future therapeutic RNA design.

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.

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.

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 define 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.

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.

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.