CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DR hi CD11c hi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR lo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach (dRNA-seq) selective for the 5' end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. We also discovered an unexpected number of approximately 60 small RNAs including the epsilon-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary transcriptomes of many living species.

Research on the discovery and characterization of small, regulatory RNAs in bacteria has exploded in recent years. These sRNAs act by base pairing with target mRNAs with which they share limited or extended complementarity, or by modulating protein activity, in some cases by mimicking other nucleic acids. Mechanistic insights into how sRNAs bind mRNAs and proteins, how they compete with each other, and how they interface with ribonucleases are active areas of discovery. Current work also has begun to illuminate how sRNAs modulate expression of distinct regulons and key transcription factors, thus integrating sRNA activity into extensive regulatory networks. In addition, the application of RNA deep sequencing has led to reports of hundreds of additional sRNA candidates in a wide swath of bacterial species. Most importantly, recent studies have served to clarify the abundance of remaining questions about how, when and why sRNA-mediated regulation is of such importance to bacterial lifestyles.

Hfq is an RNA-binding protein that is common to diverse bacterial lineages and has key roles in the control of gene expression. By facilitating the pairing of small RNAs with their target mRNAs, Hfq affects the translation and turnover rates of specific transcripts and contributes to complex post-transcriptional networks. These functions of Hfq can be attributed to its ring-like oligomeric architecture, which presents two non-equivalent binding surfaces that are capable of multiple interactions with RNA molecules. Distant homologues of Hfq occur in archaea and eukaryotes, reflecting an ancient origin for the protein family and hinting at shared functions. In this Review, we describe the salient structural and functional features of Hfq and discuss possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo.Hfq was discovered in Escherichia coli nearly half a century ago (BOX 1) and was one of the first recognized representatives of an extensive RNA-binding protein family, the members of which can be found in almost every cellular organism from all three domains of life 1 . The meta-zoan homologues of Hfq include the Sm proteins, named after the autoimmune Sm antibodies that recognize them, and the closely related Sm-like (LSm) proteins, which are also found in single-celled eukaryotes and in archaea. The characteristic feature of the collective Hfq-Sm-LSm protein family is a ring-like, multimeric quaternary architecture that supports interactions with partner macromolecules. Both Hfq and the SmLSm proteins have general roles as RNA binders that contribute to post-transcriptional regulation. The Sm-LSm proteins include central components of the mRNA-splicing machinery, scaffolds for RNA-decapping assemblies, and protective chaperones of ribosomal RNAs, small nucleolar RNAs and tRNA precursors 1 .Competing interests statement The authors declare no competing financial interests. Today, Hfq is perceived primarily as the core component of a global post-transcriptional network, in which it facilitates the short and imperfect base-pairing interactions of regulatory small RNAs (sRNAs) with trans-encoded target mRNAs. Model organisms such as E. coli or Salmonella enterica can express ~100 different sRNAs. Unlike their functional equivalents in eukaryotes -the 22-nucleotide-long microRNAs -these bacterial sRNAs are heterogeneous in size and structure. The intriguing physiological functions of Hfq and sRNAs have recently been reviewed [2][3][4][5] . FURTHER INFORMATIONThere are several general mechanisms of Hfq-mediated regulation at the levels of translation or RNA stability [2][3][4][5][6][7] , and these are summarized in FIG. 1. First, Hfq can suppress protein synthesis by aiding a cognate sRNA to bind the 5′ region of its target mRNA, thus rendering this 5′ region inaccessible for translation initiation (FIG. 1a). Conversely, Hfq can boost translation by guiding an sRNA to the 5′ region of its target mRNA in order to disrupt a secondary str...