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

On the stage of bacterial signal transduction and regulation, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has long played the part of Sleeping Beauty. c-di-GMP was first described in 1987, but only recently was it recognized that the enzymes that 'make and break' it are not only ubiquitous in the bacterial world, but are found in many species in huge numbers. As a key player in the decision between the motile planktonic and sedentary biofilm-associated bacterial 'lifestyles', c-di-GMP binds to an unprecedented range of effector components and controls diverse targets, including transcription, the activities of enzymes and larger cellular structures. This Review focuses on emerging principles of c-di-GMP signalling using selected systems in different bacteria as examples.

Principles of c-di-GMP signalling in bacteria.

▪ Abstract ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.

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RNA Editing by Adenosine Deaminases That Act on RNA

Discoveries over the past decade portend a paradigm shift in molecular biology. Evidence suggests that RNA is not only functional as a messenger between DNA and protein but also involved in the regulation of genome organization and gene expression, which is increasingly elaborate in complex organisms. Regulatory RNA seems to operate at many levels; in particular, it plays an important part in the epigenetic processes that control differentiation and development. These discoveries suggest a central role for RNA in human evolution and ontogeny. Here, we review the emergence of the previously unsuspected world of regulatory RNA from a historical perspective.

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The rise of regulatory RNA.

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.

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RNA editing with CRISPR-Cas13

Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.

/pdf/a-bacteriophage-encodes-its-own-crispr-cas-adaptive-response-2ei187q82i.pdf

A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity

RNA editing catalyzed by ADAR1 and ADAR2 involves the site-specific conversion of adenosine to inosine within imperfectly duplexed RNA. ADAR1- and ADAR2-mediated editing occurs within transcripts of glutamate receptors (GluR) in the brain and in hepatitis delta virus (HDV) RNA in the liver. Although the Q/R site within the GluR-B premessage is edited more efficiently by ADAR2 than it is by ADAR1, the converse is true for the 160 site within this same transcript. ADAR1 and ADAR2 are homologs having two common functional regions, an N-terminal doublestranded RNA-binding domain and a C-terminal deaminase domain. It is neither understood why only certain adenosines within a substrate molecule serve as targets for ADARs, nor is it known which domain of an ADAR confers its specificity for particular editing sites. To assess the importance of several aspects of RNA sequence and structure on editing, we evaluated 20 different mutated substrates, derived from four editing sites, for their ability to be edited by either ADAR1 or ADAR2. We found that when these derivatives contained an A:C mismatch at the editing site, editing by both ADARs was enhanced compared to when A:A or A:G mismatches or A:U base pairs occurred at the same site. Hence substrate recognition and/or catalysis by ADARs could involve the base that opposes the edited adenosine. In addition, by using protein chimeras in which the deaminase domains were exchanged between ADAR1 and ADAR2, we found that this domain played a dominant role in defining the substrate specificity of the resulting enzyme.

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Substrate recognition by ADAR1 and ADAR2.

Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment.

/pdf/gene-fitness-landscapes-of-vibrio-cholerae-at-important-4yfh0viygm.pdf

Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

Hepatitis delta virus (HDV) uses a host-encoded RNA-editing activity to express its two essential proteins from the same coding sequence. Adenosine deaminase that acts on RNA (ADAR)1 and ADAR2 are enzymes that catalyze such reactions, and each, when overexpressed, are capable of editing HDV RNA in vivo. However, the enzyme responsible for editing HDV RNA during replication has not been determined. Mammalian cells express two forms of ADAR1, a large form (ADAR1-L) that mainly localizes to the cytoplasm and a small form (ADAR1-S) that resides in the nucleus. Recently, we found that the specific activity of ADAR1-L within cells is much higher than that of ADAR1-S but only when the substrate can be edited in the cytoplasm. Here we observed that although both ADAR1-S and ADAR1-L were expressed throughout HDV replication, no ADAR2 could be observed at any time. Using expression vectors that individually overexpress either form of ADAR1, we found that ADAR1-S could stimulate editing during replication more efficiently. We next reduced ADAR1 levels during HDV replication. After transfection of an ADAR1-L-specific small interfering RNA (siRNA), we observed a significant loss of that protein and its associated cytoplasmic editing activity while the level of ADAR1-S remained unchanged. Transfection of this siRNA, however, did not reduce editing during HDV replication. In contrast, transfection of an siRNA that targets both forms of ADAR1 greatly reduced the expression of both proteins and potently inhibited editing during replication. We conclude that ADAR1-S edits HDV RNA during replication and that editing occurs in the nucleus.

https://www.pnas.org/content/pnas/99/23/15118.full.pdf

Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1.

Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance in Escherichia coli. We constructed a highly saturated transposon library that covered the majority of E. coli genes and promoter regions and exposed a stationary-phase culture to a lethal dose of gentamicin. Tn-Seq was performed to evaluate the survival of each mutant to gentamicin exposure. We found that the disruption of several distinct pathways affected gentamicin tolerance. We identified 105 disrupted gene/promoter regions with a more than 5-fold reduction in gentamicin tolerance and 37 genes with a more than 5-fold increased tolerance. Functional cluster analysis suggests that deficiency in motility and amino acid synthesis significantly diminished persisters tolerant to gentamicin, without changing the MIC. Amino acid auxotrophs, including serine, threonine, glutamine, and tryptophan auxotrophs, exhibit strongly decreased tolerance to gentamicin, which cannot be restored by supplying the corresponding amino acids to the culture. Interestingly, supplying these amino acids to wild-type E. coli sensitizes stationary-phase cells to gentamicin, possibly through the inhibition of amino acid synthesis. In addition, we found that the deletion of amino acid synthesis genes significantly increases gentamicin uptake in stationary phase, while the deletion of flagellar genes does not affect gentamicin uptake. We conclude that activation of motility and amino acid biosynthesis contributes to the formation of persisters tolerant to gentamicin. IMPORTANCE Persisters are responsible for the recalcitrance of chronic infections to antibiotics. The pathways of persister formation in E. coli are redundant, and our understanding of the mechanism of persister formation is incomplete. Using a highly saturated transposon insertion library, we systematically analyzed the contribution of different cellular processes to the formation of persisters tolerant to aminoglycosides. Unexpectedly, we found that activation of amino acid synthesis and motility strongly contributes to persister formation. The approach used in this study leads to an understanding of aminoglycoside tolerance and provides a general method to identify genes affecting persister formation.

https://journals.asm.org/doi/pdf/10.1128/mBio.00078-15

David W. Lazinski

Papers

A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity

Substrate recognition by ADAR1 and ADAR2.

Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1.

Genetic Basis of Persister Tolerance to Aminoglycosides in Escherichia coli