Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function—it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.American Cancer Society (Robbie Sue Mudd Kidney Cancer Research Scholar Grant RSG-13-396-01-RMC)National Institutes of Health (U.S.) (GM094303)National Institutes of Health (U.S.) (GM081399)American Cancer Society. New England Division (Ellison Foundation Postdoctoral Fellowship)American Cancer Society (Postdoctoral Fellowship PF-13-319-01-RMC)National Institutes of Health (U.S.) (Pre-doctoral Training Grant T32GM007287

riboviz: analysis and visualization of ribosome profiling datasets

Abstract Background Using high-throughput sequencing to monitor translation in vivo, ribosome profiling can provide critical insights into the dynamics and regulation of protein synthesis in a cell. Since its introduction in 2009, this technique has played a key role in driving biological discovery, and yet it requires a rigorous computational toolkit for widespread adoption. Description We have developed a database and a browser-based visualization tool, riboviz, that enables exploration and analysis of riboseq datasets. In implementation, riboviz consists of a comprehensive and flexible computational pipeline that allows the user to analyze private, unpublished datasets, along with a web application for comparison with published yeast datasets. Source code and detailed documentation are freely available from https://github.com/shahpr/RiboViz . The web-application is live at www.riboviz.org. Conclusions riboviz provides a comprehensive database and analysis and visualization tool to enable comparative analyses of ribosome-profiling datasets. This toolkit will enable both the community of systems biologists who study genome-wide ribosome profiling data and also research groups focused on individual genes to identify patterns of transcriptional and translational regulation across different organisms and conditions

Endogenous rRNA Sequence Variation Can Regulate Stress Response Gene Expression and Phenotype

Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, ReIA and ReIE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology

Identification of New Branch Points and Unconventional Introns in Saccharomyces Cerevisiae

Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5′ splice site (5′SS), branch point (BP), and 3′ splice site (3′SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BPs and 5′SSs of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We performed RNA-seq to confirm associated 3′SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3′SS choice, protein coding potential, or RNA stability, and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings show unanticipated complexity of splicing in yeast.National Science Foundation (U.S.) (Grant 0821391

Identification of new branch points and unconventional introns in Saccharomyces cerevisiae

Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5′ splice site (5′SS), branch point (BP), and 3′ splice site (3′SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BPs and 5′SSs of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We performed RNA-seq to confirm associated 3′SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3′SS choice, protein coding potential, or RNA stability, and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings show unanticipated complexity of splicing in yeast.National Science Foundation (U.S.) (Grant 0821391

Origin and differential selection of allelic variation at TAS2R16 associated with salicin bitter taste sensitivity in Africa

Bitter taste perception influences human nutrition and health, and the genetic variation underlying this trait may play a role in disease susceptibility. To better understand the genetic architecture and patterns of phenotypic variability of bitter taste perception, we sequenced a 996 bp region, encompassing the coding exon of TAS2R16, a bitter taste receptor gene, in 595 individuals from 74 African populations and in 94 non-Africans from 11 populations. We also performed genotype-phenotype association analyses of threshold levels of sensitivity to salicin, a bitter anti-inflammatory compound, in 296 individuals from Central and East Africa. In addition, we characterized TAS2R16 mutants in vitro to investigate the effects of polymorphic loci identified at this locus on receptor function. Here, we report striking signatures of positive selection, including significant Fay and Wu's H statistics predominantly in East Africa, indicating strong local adaptation and greater genetic structure among African populations than expected under neutrality. Furthermore, we observed a "star-like" phylogeny for haplotypes with the derived allele at polymorphic site 516 associated with increased bitter taste perception that is consistent with a model of selection for "high-sensitivity" variation. In contrast, haplotypes carrying the "low-sensitivity" ancestral allele at site 516 showed evidence of strong purifying selection. We also demonstrated, for the first time, the functional effect of nonsynonymous variation at site 516 on salicin phenotypic variance in vivo in diverse Africans and showed that most other nonsynonymous substitutions have weak or no effect on cell surface expression in vitro, suggesting that one main polymorphism at TAS2R16 influences salicin recognition. Additionally, we detected geographic differences in levels of bitter taste perception in Africa not previously reported and infer an East African origin for high salicin sensitivity in human populations