Hrp1/Nab4 is an essential nuclear RNA-binding protein that was first identified in the yeast Saccharomyces cerevisiae as a cleavage and polyadenylation factor for mRNAs, CF1B, but was later shown to promote termination of some short, noncoding transcripts via the "NNS" termination pathway. Hrp1 binds (UA)n repeats found in both mRNA 3'-UTRs and short noncoding RNA terminators, but its function in 3'-end formation is not fully understood. Our past microarray transcriptome analysis of the heat-sensitive hrp1-7 allele suggested Hrp1 functions in antitermination of RNA polymerase II (RNAP II) on protein-coding genes. The hrp1-7 allele has four substitutions and one, M191T, was shown to be primarily responsible for NNS terminator readthrough. Here we show that Hrp1-7 protein has a three-fold and Hrp1-M191T a 1.5-fold decreased affinity for (UA)4 in vitro. We used nanopore direct RNA sequencing to assess the transcriptome-wide effects of hrp1-7 and hrp1-M191T, which identified new examples of Hrp1-dependent small nucleolar RNA terminators and mRNA attenuators (regulatory terminators in the 5'-UTR and ORF). For some genes, including HRP1, attenuated transcript reads outnumber the corresponding mRNA reads in exponentially growing wildtype cells. We also observed widespread changes in mRNA polyA site selection. In the hrp1-M191T strain the polyA site shifts are mostly downstream, while in the hrp1-7 strain upstream and downstream shifts are more similar in frequency. Our results are consistent with a model in which one or more of the four substitutions in hrp1-7 weaken the affinity of Hrp1 for the RNAP II elongation complex, promoting premature termination, while others interfere with its recognition of terminator sequences, promoting terminator readthrough.

The partners of an ecological association tend to copy the biological system of their hosts. We hypothesized that microorganisms, particularly pathogens, have acquired the ability to express short peptides (pathoPEPs) homologous to host miPEPs, thus modulating the expression of the corresponding host microRNA (miRNA) and the function of miRNA-targeted genes. The pathosystem involving interactions between Brassica napus and its pathogen Plasmodiophora brassicae was studied. The data set contains RNA-seq and ribosomal profiling from two plant genotypes inoculated with one pathogen isolate. Using in silico analysis, we identified three putative pathoPEPs produced by P. brassicae and their targeted plant miRNA genes. A link between the level of infection of B. napus by P. brassicae and the expression of pathoPEPs and their targeted miRNA genes was found, with the expression of the latter two being inversely related. Finally, we identified differential expression and translation of genes predicted to be targets of pathoPEP-regulated miRNAs. These genes, involved in auxin pathway, immune defense, root architecture, or carbohydrate metabolism, are thought to enable P. brassicae, through its pathoPEPs, to hijack plant's metabolic pathways (e.g., hormonal pathways, sugar synthesis, root morphology), thereby facilitating its invasion. Using in silico approaches, the involvement of miPEPs from this host-pathogen system as a host post-transcriptional regulatory pathway is described herein for the first time.

Angiosarcoma (AS) is a rare and aggressive tumor arising within the endothelium, characterized by a high metastatic rate and poor prognosis. Our prior work established that endothelial loss of Dicer1, a key enzyme in microRNA (miRNA) processing, drives AS formation in mice, indicating a tumor suppressive role for miRNAs in tumorigenesis. Here, we corroborated this hypothesis by generating a novel conditional knockout model targeting Dgcr8, a core component of the microprocessor complex required for pri-miRNA processing. Conditional deletion of Dgcr8 phenocopies Dicer1 loss, resulting in spontaneous AS formation and global loss of mature miRNAs. We further demonstrate that treatment with enoxacin (ENX), a repurposed antibiotic known to enhance miRNA processing, reduces viability, migration, and clonogenicity of AS cells. ENX increases the abundance of tumor-suppressive miRNAs and downregulates oncogenic pathways, including pathways related to cell cycle progression, angiogenesis, and cell migration. These results establish the essential role of miRNA biogenesis in suppressing AS and reveal a pharmacologically targetable vulnerability via ENX-mediated enhancement of miRNA expression in tumors.

RNase L is an endonuclease that responds to infections by cleaving most host- and pathogen-derived single-stranded RNAs. This widespread RNA cleavage can lead to death of the infected cell via the ribotoxic stress response (RSR). An ongoing challenge is to understand how RNase L's endonuclease activity triggers cell death to benefit the host. To address this question, we used nanopore-based long-read sequencing to show that 3' mRNA fragments in the cell were not fully degraded after RNase L activation and that these fragments were translated by ribosomes. We further asked whether ribosomes on mRNA fragments stall when they reach 3' ends created by RNase L. We used ribosome profiling to capture footprints protected by these ribosomes, which can be identified by their short length (15-18 nt). We found that RNase L activation increased the number of stalled ribosomes at RNase L cleavage sites. Loss of the ribosome rescue factor PELO increased the number of short footprints derived from stalled ribosomes and augmented the RSR. Our work therefore establishes a role for fragmented mRNA in causing ribosome stalling that promotes innate immunity via the RSR.

EXOSC10 is a conserved 3'-5' exoribonuclease involved in processing ribosomal RNAs and degrading coding and noncoding transcripts as a catalytic subunit of the nuclear RNA exosome and in cooperation with cofactors. The protein is posttranslationally modified and shuttles between the nucleolus and the nucleus in response to oxygen deprivation in a process that involves sumoylation. EXOSC10 is of medical interest because its activity is inhibited by the anticancer drug 5-fluorouracil, which interferes with DNA replication and RNA-dependent processes. Moreover, high expression of EXOSC10 in certain somatic tumors is associated with patient survival. We discuss global and tissue-specific deletion experiments in the mouse, assess the protein's clinical relevance as a prognostic cancer biomarker in the context of human genomics data for normal versus malignant tissues, and explore EXOSC10's transcriptional regulatory network.

Detection of foreign RNAs is a crucial activation step for innate immunity pathways in response to viral infections. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic RNA sensor that triggers type I and III interferon (IFN) expression and activates the antiviral response during RNA virus infections. The activating ligand for RIG-I has been shown to be 5'-triphosphated, blunt-ended, double-stranded (ds)RNA, but questions remain on the impact of other RNA motifs on RIG-I activation. Here we show that immune-activating copy-back viral genomes (cbVGs) contain RNA stem-loops away from the 5' end of the RNA that enhance RIG-I signaling and IFN expression. Importantly, the sequence of the terminal loops of the activating motifs impacts the strength of IFN expression. Additionally, we show that synthetic versions of these cbVG-derived stem-loops trigger innate immune responses in mice, demonstrating their potential as immunostimulants in vivo.

MicroRNAs (miRNAs) are key regulators of gene expression in both health and disease. Their expression and regulatory functions are highly complex and spatiotemporally organized within tissues. In recent years, spatial transcriptomics has made significant progress in quantifying RNA expression at subcellular resolution in tissue sections. However, no current method can quantify miRNAs and their target 3' untranslated regions (3'UTRs) in space simultaneously. Furthermore, although 3'UTRs harbor critical miRNA target sites, 3'UTR isoform variation in space is largely unexplored. In this review, we discuss the role of miRNA-mediated regulation. We focus on neurodevelopment and neuronal function, where miRNAs and 3'UTRs have particularly complex and important functions. We summarize current experimental and computational approaches for spatial quantification of miRNAs and 3'UTRs, highlight existing challenges, and propose strategies for future research.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape strategies include general inhibition of host gene expression referred to as host shutoff. Viral nonstructural protein 1 (Nsp1) is the main host shutoff factor that blocks protein translation and induces messenger RNA (mRNA) cleavage and degradation. Viral mRNAs are resistant to the translation shutoff and cleavage induced by Nsp1, and the 5' leader sequence present in all viral mRNAs has been shown to confer resistance. However, the exact molecular mechanism for escape from Nsp1 host shutoff has not been demonstrated. In our previous work, we analyzed the effects of Nsp1 on the expression and function of cellular proteins important for stress granule formation. We discovered that the host transcript for the TIA1 cytotoxic granule-associated RNA-binding protein-like 1 (TIAL1, commonly referred to as TIAR) is resistant to SARS-CoV-2 Nsp1 host shutoff. In this work, using reporter shutoff assays, we examined sequence and structural features of the TIAR 5' untranslated region (UTR) and discovered that the first 23 nt of the TIAR transcript are both necessary and sufficient to confer resistance to the Nsp1. Furthermore, our work revealed that the lack of guanosines within a window of 10-18 nt downstream from the 5' end is a defining feature of Nsp1-resistant transcripts shared between the SARS-CoV-2 leader sequence and the TIAR 5' UTR. Our findings are consistent with the model in which sequence features of 5' UTRs, rather than their secondary structure, confer resistance to Nsp1 host shutoff to both viral and cellular mRNAs.

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.