Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

骨髄異形成症候群のgenome-wide analysis

RNA-binding proteins (RBPs) are central to most if not all cellular processes, dictating the fate of virtually all RNA molecules in the cell. Starting with pioneering work on ribosomal proteins, studies of bacterial RBPs have paved the way for molecular studies of RNA–protein interactions. Work over the years has identified major RBPs that act on cellular transcripts at the various stages of bacterial gene expression and that enable their integration into post-transcriptional networks that also comprise small non-coding RNAs. Bacterial RBP research has now entered a new era in which RNA sequencing-based methods permit mapping of RBP activity in a truly global manner in vivo. Moreover, the soaring interest in understudied members of host-associated microbiota and environmental communities is likely to unveil new RBPs and to greatly expand our knowledge of RNA–protein interactions in bacteria.

/pdf/rna-binding-proteins-in-bacteria-4h5r6wguh0.pdf

RNA-binding proteins in bacteria

RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles

The last few decades have led to an explosion in our understanding of the major roles that small regulatory RNAs (sRNAs) play in regulatory circuits and the responses to stress in many bacterial species. Much of the foundational work was carried out with Escherichia coli and Salmonella enterica serovar Typhimurium. The studies of these organisms provided an overview of how the sRNAs function and their impact on bacterial physiology, serving as a blueprint for sRNA biology in many other prokaryotes. They also led to the development of new technologies. In this chapter, we first summarize how these sRNAs were identified, defining them in the process. We discuss how they are regulated and how they act and provide selected examples of their roles in regulatory circuits and the consequences of this regulation. Throughout, we summarize the methodologies that were developed to identify and study the regulatory RNAs, most of which are applicable to other bacteria. Newly updated databases of the known sRNAs in E. coli K-12 and S. enterica Typhimurium SL1344 serve as a reference point for much of the discussion and, hopefully, as a resource for readers and for future experiments to address open questions raised in this review.

Trans-Acting Small RNAs and Their Effects on Gene Expression in Escherichia coli and Salmonella enterica.

Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3' Ends.

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks Yet, there has been no global census of RNA/protein complexes in this model species of microbiology Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E coli transcripts and ~49% of the proteins These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species

https://biorxiv.org/content/10.1101/2020.06.29.177014v1.full.pdf

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA-sequencing (MAPS), we identify PinT ligands in bacteria under in-vitro conditions mimicking specific stages of the infection cycle, and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually preventing actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.

/pdf/maps-integrates-overlooked-regulation-of-actin-targeting-57sfif88eh.pdf

MAPS integrates overlooked regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT

The Gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, little is known about the bacterium’s transcriptome architecture and mechanisms of post-transcriptional control. Here, we have applied a suite of RNA-centric techniques, including transcription start site mapping, transcription termination mapping and Hfq RIP-seq, to generate a single-nucleotide resolution RNA map of C. difficile 630. Our transcriptome annotation provides information about 5’ and 3’ untranslated regions, operon structures and non-coding regulators, including 42 sRNAs. These transcriptome data are accessible via an open-access browser called ‘Clost-Base’. Our results indicate functionality of many conserved riboswitches and predict novel cis-regulatory elements upstream of MDR-type ABC transporters and transcriptional regulators. Recent studies have revealed a role of sRNA-based regulation in several Gram-positive bacteria but their involvement with the RNA-binding protein Hfq remains controversial. Here, sequencing the RNA ligands of Hfq reveals in vivo association of many sRNAs along with hundreds of potential target mRNAs in C. difficile providing evidence for a global role of Hfq in post-transcriptional regulation in a Gram-positive bacterium. Through integration of Hfq-bound transcripts and computational approaches we predict regulated target mRNAs for the novel sRNA AtcS encoding several adhesins and the conserved oligopeptide transporter oppB that influences sporulation initiation in C. difficile. Overall, these findings provide a potential mechanistic explanation for increased biofilm formation and sporulation in an hfq deletion strain and lay the foundation for understanding clostridial ribo regulation with implications for the infection process.

/pdf/an-rna-centric-global-view-of-clostridioides-difficile-4b0hzrd1vs.pdf

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important Gram-positive bacterium

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Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3' Ends.

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

MAPS integrates overlooked regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important Gram-positive bacterium