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Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity of Mammalian Proteomes

ABSTRACT: The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep-sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and novel translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a new class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes.

ORGANISM(S): Mus musculus

PROVIDER: GSE30839 | GEO | 2011/11/03

SECONDARY ACCESSION(S): PRJNA144149

REPOSITORIES: GEO

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Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity of Mammalian Proteomes

Project description:The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep-sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and novel translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a new class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes. Examination of translation in mouse embryonic stem cells and during differentiation into embryoid bodies

2011-11-03 | E-GEOD-30839 | biostudies-arrayexpress

Systems biology decodes herpes simplex virus 1

Project description:Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, the 84 genes it was thought to encode have been intensively studied. Here, we unravel the full complement of viral transcription and translation during lytic infection with base-pair resolution by computational integration of multi-omics data. This includes a total of 201 viral transcripts and 296 open reading frames (ORFs), comprising all known large ORFs, 55 large novel ORFs including a novel spliced ORF in the ICP0 locus that initiates from a non-AUG start codon and a novel strongly translated ORF in the ICP34.5 locus as well as multiple short ORFs. By defining viral gene modules, we link translation of ORFs to the expression of transcript isoforms. This explained translation of the vast majority of viral ORFs as well as N-terminal extensions and truncations thereof. We show that N-terminal extensions initiating from non-AUG start codons govern subcellular protein localization and packaging of key viral regulators and structural proteins. This work has great implications for future functional studies, vaccine design and novel oncolytic therapies.

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Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells

Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells. Ribosome profiling or stranded RNAseq (ribominus) with murine embryonic stem cells treated with either DMD-pateamineA, Puromycin, Harringtonine or vehicle (no drug control). Two replicates per condition.

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The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions

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

Project description:Meiosis is a complex developmental process that generates haploid cells from diploid progenitors. We measured mRNA abundance and protein production through yeast sporulation and found strong temporal control for most genes, achieved through both mRNA levels and translational regulation. Monitoring the timing of protein production revealed novel factors involved in recombination and helped to illuminate the molecular basis of the broad restructuring of meiotic cells. We also found a strong increase in noncanonical translation at short open reading frames (sORFs) on unannnotated transcripts and upstream regions of known transcripts (uORFs). Ribosome occupancy at near-cognate uORFs was associated with more efficient ORF translation; while some AUG uORFs, often on regulated leader extensions, acted comptetitively. This work reveals a pervasive role for meiotic translational control and great complexity in genomic coding. Fine mapping of gene expression through meiosis reveals extensive regulation of protein synthesis and widespread non-canonical translation.

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eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide

Project description:The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at the NCCs initiating N-terminal extensions on GRS1 and ALA1 mRNAs, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

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Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors

Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.

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Mechanisms of stop codon readthrough mitigation reveal principles of GCN1 mediated translational quality control

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