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High-resolution view of the yeast meiotic program revealed by ribosome profiling.

ABSTRACT: Meiosis is a complex developmental process that generates haploid cells from diploid progenitors. We measured messenger RNA (mRNA) abundance and protein production through the yeast meiotic sporulation program and found strong, stage-specific expression for most genes, achieved through control of both mRNA levels and translational efficiency. Monitoring of protein production timing revealed uncharacterized recombination factors and extensive organellar remodeling. Meiotic translation is also shifted toward noncanonical sites, including short open reading frames (ORFs) on unannnotated transcripts and upstream regions of known transcripts (uORFs). Ribosome occupancy at near-cognate uORFs was associated with more efficient ORF translation; by contrast, some AUG uORFs, often exposed by regulated 5' leader extensions, acted competitively. This work reveals pervasive translational control in meiosis and helps to illuminate the molecular basis of the broad restructuring of meiotic cells.

SUBMITTER: Brar GA

PROVIDER: S-EPMC3414261 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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High-resolution view of the yeast meiotic program revealed by ribosome profiling.

Brar Gloria A GA Yassour Moran M Friedman Nir N Regev Aviv A Ingolia Nicholas T NT Weissman Jonathan S JS

Science (New York, N.Y.) 20111222 6068

Meiosis is a complex developmental process that generates haploid cells from diploid progenitors. We measured messenger RNA (mRNA) abundance and protein production through the yeast meiotic sporulation program and found strong, stage-specific expression for most genes, achieved through control of both mRNA levels and translational efficiency. Monitoring of protein production timing revealed uncharacterized recombination factors and extensive organellar remodeling. Meiotic translation is also shi ...[more]

PMID: 22194413

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Project description:We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.

Project description:Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

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High-resolution view of the yeast meiotic program revealed by ribosome profiling.

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High-resolution view of the yeast meiotic program revealed by ribosome profiling.

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