Redefining the translational status of 80S monosomes
ABSTRACT: Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs. We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.
Project description:Translational profiling of mouse cardiac tissue treated with 25mg/kg DMNQ in 10 ml/kg arachis oil over an acute time course (0.5-120 hours) compared to time matched control animals treated with 10ml/kg saline Two colour microarrays with time matched controls vs 25mg/kg DMNQ cardiac tissue. Before microarray analysis RNA separated on a sucrose density gradient into those mRNAs activitly undergoing translation (polysomes) and those not (monosomes) with control monosomes and treated monosomes on one set of arrays, and polysome control and polysome treated on another set of microarrays. The normalized Log2 of the monosomes was subtracted from the respective Log2 of the polysomes (on Series record). Time points studied were 0.5, 1, 2, 12, 24 and 120 hours following dosing, biological replicates n=3 independent animals at each time point, technical replicates (reverse labelling) n<1. One array printed onto two slides (A and B), one replicate per array.
Project description:Translational profiling of mouse cardiac tissue treated with 15mg/kg doxorubicin in 10 ml/kg saline over an acute time course (0.5-120 hours) compared to time matched control animals treated with 10ml/kg saline. Two colour microarrays with time matched controls vs 15mg/kg doxorubicin cardiac tissue. Before microarray analysis, RNA is separated on a sucrose density gradient into those mRNAs activity undergoing translation (polysomes) and those not (monosomes) with control monosomes and treated monosomes on one set of arrays, and polysome control and polysome treated on another set of microarrays. Thealized Log2 of the monosomes was subtracted from the respective Log2 of the polysomes (on Series record). Time points studied were 0.5, 1, 2, 12, 24 and 120 hours following dosing, biological replicates n=3 independent animals at each time point, technical replicates (reverse labelling) n<1. One array printed onto two slides (A and B), one replicate per array.
Project description:In order to deal with their huge volume and complex morphology, neurons localize mRNAs and ribosomes near synapses to produce proteins locally. A relative paucity of polyribosomes (considered the active sites of translation) detected in electron micrographs of neuronal processes (axons and dendrites), however, has suggested a rather limited capacity for local protein synthesis. Polysome profiling together with ribosome footprinting of microdissected synaptic regions revealed that a surprisingly high number of dendritic and/or axonal transcripts were predominantly associated with monosomes (single ribosomes). Contrary to prevailing views, the neuronal monosomes were in the process of active protein synthesis (e.g. they exhibited elongation). Most mRNAs showed a similar translational status in the somata and neuropil, but some transcripts exhibited differential ribosome occupancy in the compartments. Strikingly, monosome-preferring transcripts often encoded high-abundance synaptic proteins. These data suggest a significant contribution of monosome translation to the maintenance of the local neuronal proteome. This mode of translation can presumably solve some of restricted space issues (given the large size of polysomes) and also increase the diversity of proteins made from a limited number of ribosomes available in dendrites and axons.
Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation
Project description:Covalent nucleotide modifications in noncoding RNAs such as tRNAs affect a plethora of biological processes, with new functions continuing to be discovered for even well-known tRNA modifications. To systematically compare the functions of a large set of ncRNA modifications in gene regulation, we carried out ribosome profiling and RNA-Seq in budding yeast for 57 nonessential genes involved in tRNA modification. Overall design: Yeast strains were treated with cycloheximide and proceeded to ribosome profiling. For ribosome-protected footprints (RPF), 80S monosome fractions were isolated after RNase I treatment, and 27-34 nt RPF were isolated by denaturing PAGE. For RNA-Seq, total RNA was depleted of rRNA using Ribo-Zero, followed by zinc-based fragmentation. RNA fragments and RPF are constructed into Illumina deep-sequencing libraries by RNA 3’ adaptor ligation and cDNA circularization. Barcoded libraries were sequenced on an Illumina NextSeq 500. Raw fastq reads were de-multiplexed and removed of adaptor sequence. RPF reads were mapped to S. cerevisiae rDNA and the mapping reads were discarded. The remaining RPF reads and RNA-seq reads were mapped to sacCer3 genome. Uniquely mapping reads in length of 27-34 nt (RPF) or 27 nt (RNA-seq) were quantified as RPKM.
Project description:Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be non-coding, including 5' UTRs and lncRNAs. Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here we show hallmarks of translation in these footprints: co-purification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts to understand how cells manage and exploit its consequences. Ribosome profiling to verify that true ribosome footprints shift in response to different elongation inhibitors (CHX vs Emetine) and co-purify with an affinity-tagged large ribosomal subunit (bound vs input)
Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Monitor the translational status of transcriptome in mammalian cells under different conditions
Project description:Cytoplasmic degradation of eukaryotic mRNAs in 3’ to 5’ direction is catalyzed by the exosome complex together with the Ski complex and, in the yeast Saccharomyces cerevisiae, the Ski7 protein. This exosome-Ski-system also degrades ribosome-associated aberrant mRNAs lacking a stop codon, a pathway called non-stop-mRNA decay (NSD). However, the interplay between the Ski components and the ribosome is unknown. Here, we report that the Ski complex can associate with ribosomes in vitro and in vivo independently of Ski7. Ribosome profiling suggests that this association is important for NSD but also for general mRNA turnover. A cryo-electron microscopy structure reveals that the Ski complex binds near the mRNA entry site of the 40S subunit, facilitating the threading of the mRNA into the Ski2 helicase. Collectively, these results demonstrate an unanticipated role for the Ski complex in mRNA decay of ribosome-associated mRNAs, mediated by a direct interaction with the translation machinery. Overall design: Ribosome profiling of 80S monosomes and in vivo pullouts of Ski complex associated 80S ribosomes. In vivo pullouts were performed using a yeast strain expressing C-terminally TAP (tandem affinity purification) tagged Ski3. Control mRNA from the total cell lysate and pull-down mRNA from the pullouts were also sequenced respectively.
Project description:We performed ribosome profiling of wild-type and loc1- cells. We found that approximately 1500 genes exhibit increased translational efficiencies in the absence of Loc1, indicating that Loc1 is required for their translational repression. Examination of translational efficiencies by comparing ribosome footprints and mRNA abundances in two different cell lines.