Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.

In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.

Pre-mRNA-splicing and adenosine to inosine (A-to-I) RNA-editing occur mostly cotranscriptionally. During A-to-I editing, a genomically encoded adenosine is deaminated to inosine by adenosine deaminases acting on RNA (ADARs). Editing-competent stems are frequently formed between exons and introns. Consistently, studies using reporter assays have shown that splicing efficiency can affect editing levels. Here, we use Nascent-seq and identify ∼90,000 novel A-to-I editing events in the mouse brain transcriptome. Most novel sites are located in intronic regions. Unlike previously assumed, we show that both ADAR (ADAR1) and ADARB1 (ADAR2) can edit repeat elements and regular transcripts to the same extent. We find that inhibition of splicing primarily increases editing levels at hundreds of sites, suggesting that reduced splicing efficiency extends the exposure of intronic and exonic sequences to ADAR enzymes. Lack of splicing factors NOVA1 or NOVA2 changes global editing levels, demonstrating that alternative splicing factors can modulate RNA editing. Finally, we show that intron retention rates correlate with editing levels across different brain tissues. We therefore demonstrate that splicing efficiency is a major factor controlling tissue-specific differences in editing levels.