Biogenesis of mature piRNAs in Caenorhabditis elegans is dependent on orchestration between multiple ribonucleases and PRG-1

Piwi-interacting RNAs (piRNAs) are an animal-specific class of germline-enriched small non-coding RNAs that shape transcriptome, as well as ensure genomic integrity and fertility by regulating transposons and other selfish genetic elements. In Caenorhabditis elegans mature piRNAs are 21-nucleotides long, begin with a monophosphorylated uridine, and they associate with PRG-1 to form piRISCs that scan the transcriptome for non-self sequences. However, these piRNAs are born as longer 5-capped transcripts, where PARN-1, a 3-5 exoribonuclease, contributes to the formation of the mature 3-end. But, till date, the 5-processing events remain elusive. We demonstrate that the recently identified endoribonuclease activity of XRN-2 is involved in the processing of the 5-end of precursor piRNAs in worms. Depletion of XRN-2 results in reduced mature piRNA levels, with concomitant increase in levels of the 5-capped precursors. We also reveal that the piRNAs born as longer precursor molecules (>60 nt), prior to 5-end processing, undergo ENDU-1-mediated endoribonucleolytic processing of their 3-ends. Our in vitro RNA-protein interaction studies unravel the mechanistic interactions between XRN-2 and PRG-1 towards the formation of mature 5-ends of piRNAs. In vivo experiments employing prg-1 mutant worms indicate that XRN-2 has the potential to perform clearance of precursors that are not bound and protected by PRG-1. Finally, we also demonstrate that XRN-2 is not only important for the generation of mature piRNAs and piRNA-dependent endo-siRNAs, but through yet unknown pathways, it also affects piRNA-independent endo-siRNAs that shape transcriptome, as well as contribute to genomic integrity via regulation of transposable elements.

Rapid Identification of Secondary Structure and Binding Site Residues in an Intrinsically Disordered Protein Segment

Mycobacterium tuberculosis harbours nine toxin-antitoxin (TA) systems of the MazEF family. MazEF TA modules are of immense importance due to the perceived role of the MazF toxin in M. tuberculosis persistence and disease. The MazE antitoxin has a disordered C-terminal domain that binds the toxin, MazF and neutralizes its endoribonuclease activity. However, the structure of most MazEF TA complexes remains unsolved till date, obscuring structural and functional information about the antitoxins. We present a facile method to identify toxin binding residues on the disordered antitoxin. Charged residue scanning mutagenesis was used to screen a yeast surface displayed MazE6 antitoxin library against its purified cognate partner, the MazF6 toxin. Binding residues were deciphered by probing the relative reduction in binding to the ligand by flow cytometry. We have used this to identify putative antitoxin interface residues and local structure attained by the antitoxin upon interaction in the MazEF6 TA system and the same methodology is readily applicable to other intrinsically disordered protein regions.

Positively Charged Amino Acids in the Pestiviral Erns Control Cell Entry, Endoribonuclease Activity and Innate Immune Evasion

The genus Pestivirus, family Flaviviridae, includes four economically important viruses of livestock, i.e., bovine viral diarrhea virus-1 (BVDV-1) and -2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). Erns and Npro, both expressed uniquely by pestiviruses, counteract the host’s innate immune defense by interfering with the induction of interferon (IFN) synthesis. The structural envelope protein Erns also exists in a soluble form and, by its endoribonuclease activity, degrades immunostimulatory RNA prior to their activation of pattern recognition receptors. Here, we show that at least three out of four positively-charged residues in the C-terminal glycosaminoglycan (GAG)-binding site of BVDV-Erns are required for efficient cell entry, and that a positively charged region more upstream is not involved in cell entry but rather in RNA-binding. Moreover, the C-terminal domain on its own determines intracellular targeting, as GFP fused to the C-terminal amino acids of Erns was found at the same compartments as wt Erns. In summary, RNase activity and uptake into cells are both required for Erns to act as an IFN antagonist, and the C-terminal amphipathic helix containing the GAG-binding site determines the efficiency of cell entry and its intracellular localization.

Global analysis of the specificities and targets of endoribonucleases from E. coli toxin-antitoxin systems

Toxin-antitoxin systems are widely distributed genetic modules typically featuring toxins that can inhibit bacterial growth and antitoxins that can reverse inhibition. Although Escherichia coli encodes 11 toxins with known or putative endoribonuclease activity, the target of most of these toxins remain poorly characterized. Using a new RNA-seq pipeline that enables the mapping and quantification of RNA cleavage with single-nucleotide resolution, we characterize the targets and specificities of 9 endoribonuclease toxins from E. coli. We find that these toxins use low-information cleavage motifs to cut a significant proportion of mRNAs in E. coli, but not tRNAs or the rRNAs from mature ribosomes. However, all the toxins, including those that are ribosome-dependent and cleave only translated RNA, inhibit ribosome biogenesis. This inhibition likely results from the cleavage of ribosomal protein transcripts, which disrupts the stoichiometry and biogenesis of new ribosomes and causes the accumulation of aberrant ribosome precursors. Collectively, our results provide a comprehensive, global analysis of endoribonuclease-based toxin-antitoxin systems in E. coli and support the conclusion that, despite their diversity, each disrupts translation and ribosome biogenesis.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp15 Endoribonuclease

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered the economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

Endoribonuclease activity of XRN-2 is critical for RNA metabolism and survival of Caenorhabditis elegans

microRNAs (miRNAs) are known to regulate a vast majority of the eukaryotic genes by post-transcriptional means, and multiple nucleases play critical roles in the biogenesis and turnover of these regulators. A number of studies have indicated that turnover is important for determining the abundance of miRNAs, and thus, in turn govern their functionality. Recent research in Caenorhabditis elegans has revealed an ATP-independent endoribonuclease activity of the ‘miRNase’-XRN-2. Here, we report the characterization of this new enzymatic activity of the fundamentally important XRN-2, and show that it is critical for miRNA turnover and survival of quiescent dauer worms. The dual enzymatic activity of XRN-2 capacitates the mechanism of miRNA turnover to be dynamic, which might confer adaptive advantage to the organism. In continuously growing worms, this new enzymatic activity does not act on miRNAs, but it is important for the generation of mature ribosomal RNAs, which in turn is critical for translation, and thus indispensable for the survival of worms.

The secreted endoribonuclease ENDU-2 from the soma protects germline immortality in C. elegans

Multicellular organisms coordinate tissue specific responses to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, recent reports implicated release of small RNAs in regulating gene expression across tissue boundaries. Here, we show that the conserved poly-U specific endoribonuclease ENDU-2 in C. elegans is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 regulates gene expression across tissue boundaries in response to temperature alterations and contributes to maintenance of stem cell immortality, probably via retaining a stem cell specific program of gene expression.

The ADP-binding kinase region of Ire1 directly contributes to its responsiveness to endoplasmic reticulum stress

Upon endoplasmic-reticulum (ER) stress, the ER-located transmembrane protein, Ire1, is autophosphorylated and acts as an endoribonuclease to trigger the unfolded protein response (UPR). Previous biochemical studies have shown that Ire1 exhibits strong endoribonuclease activity when its cytosolic kinase region captures ADP. Here, we asked how this event contributes to the regulation of Ire1 activity. At the beginning of this study, we obtained a luminal-domain mutant of Saccharomyces cerevisiae Ire1, deltaIdeltaIIIdeltaV/Y225H Ire1, which is deduced to be controlled by none of the luminal-side regulatory events. ER-stress responsiveness of deltaIdeltaIIIdeltaV/Y225H Ire1 was largely compromised by a further mutation on the kinase region, D797N/K799N, which allows Ire1 to be activated without capturing ADP. Therefore, in addition to the ER-luminal domain of Ire1, which monitors ER conditions, the kinase region is directly involved in the ER-stress responsiveness of Ire1. We propose that potent ER stress harms cells’ “vividness”, increasing the cytosolic ADP/ATP ratio, and eventually strongly activates Ire1. This mechanism seems to contribute to the suppression of inappropriately potent UPR under weak ER-stress conditions.