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Cold Spring Harbor Perspectives in... Dec 2018This review summarizes our current understanding of the major pathway for the initiation phase of protein synthesis in eukaryotic cells, with a focus on recent advances.... (Review)
Review
This review summarizes our current understanding of the major pathway for the initiation phase of protein synthesis in eukaryotic cells, with a focus on recent advances. We describe the major scanning or messenger RNA (mRNA) mG cap-dependent mechanism, which is a highly coordinated and stepwise regulated process that requires the combined action of at least 12 distinct translation factors with initiator transfer RNA (tRNA), ribosomes, and mRNAs. We limit our review to studies involving either mammalian or budding yeast cells and factors, as these represent the two best-studied experimental systems, and only include a reference to other organisms where particular insight has been gained. We close with a brief description of what we feel are some of the major unknowns in eukaryotic initiation.
Topics: Animals; Eukaryotic Cells; Peptide Chain Initiation, Translational; Protein Biosynthesis
PubMed: 29735639
DOI: 10.1101/cshperspect.a033092 -
Science (New York, N.Y.) Apr 2009Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a...
Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon 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 in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.
Topics: 5' Untranslated Regions; Codon; Gene Library; Genome, Fungal; Introns; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA, Fungal; RNA, Messenger; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Analysis, DNA
PubMed: 19213877
DOI: 10.1126/science.1168978 -
Journal of Molecular Cell Biology Oct 2019Most eukaryotic mRNAs are translated in a cap-dependent fashion; however, under stress conditions, the cap-independent translation driven by internal ribosomal entry... (Review)
Review
Most eukaryotic mRNAs are translated in a cap-dependent fashion; however, under stress conditions, the cap-independent translation driven by internal ribosomal entry sites (IRESs) can serve as an alternative mechanism for protein production. Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trans-acting factors. Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood, the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities. A recent screen identified > 1000 putative IRESs from viral and human mRNAs, expanding the scope and mechanism for cap-independent translation. Additionally, a large number of circular RNAs lacking free ends were identified in eukaryotic cells, many of which are found to be translated through IRESs. These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA, implying a hidden proteome produced from cap-independent translation.
Topics: Humans; Internal Ribosome Entry Sites; Models, Biological; Protein Biosynthesis; Proteome; Ribosomes
PubMed: 31504667
DOI: 10.1093/jmcb/mjz091 -
Cold Spring Harbor Perspectives in... May 2019Stress granules (SGs) and processing bodies (PBs) are non-membrane-enclosed RNA granules that dynamically sequester translationally inactive messenger ribonucleoprotein... (Review)
Review
Stress granules (SGs) and processing bodies (PBs) are non-membrane-enclosed RNA granules that dynamically sequester translationally inactive messenger ribonucleoprotein particles (mRNPs) into compartments that are distinct from the surrounding cytoplasm. mRNP remodeling, silencing, and/or storage involves the dynamic partitioning of closed-loop polyadenylated mRNPs into SGs, or the sequestration of deadenylated, linear mRNPs into PBs. SGs form when stress-activated pathways stall translation initiation but allow elongation and termination to occur normally, resulting in a sudden excess of mRNPs that are spatially condensed into discrete foci by protein:protein, protein:RNA, and RNA:RNA interactions. In contrast, PBs can exist in the absence of stress, when specific factors promote mRNA deadenylation, condensation, and sequestration from the translational machinery. The formation and dissolution of SGs and PBs reflect changes in messenger RNA (mRNA) metabolism and allow cells to modulate the proteome and/or mediate life or death decisions during changing environmental conditions.
Topics: Animals; Cytoplasmic Granules; Gene Expression Regulation; Protein Biosynthesis; Ribonucleoproteins
PubMed: 30082464
DOI: 10.1101/cshperspect.a032813 -
Nature Protocols Jul 2012Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of...
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. In addition, 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 pretreating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5-7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis require a further 4-5 days.
Topics: Animals; Base Sequence; Gene Library; Harringtonines; Humans; Molecular Sequence Data; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; Ribonucleases; Ribosomes; Saccharomyces cerevisiae; Sequence Analysis, RNA; Transcriptome
PubMed: 22836135
DOI: 10.1038/nprot.2012.086 -
Cell Dec 2020Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins....
Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.
Topics: Activating Transcription Factor 4; Cytoplasmic Granules; Cytosol; HeLa Cells; Humans; Open Reading Frames; Protein Biosynthesis; RNA Transport; RNA, Messenger; Single Molecule Imaging; Stress, Physiological
PubMed: 33308477
DOI: 10.1016/j.cell.2020.11.010 -
Cell May 2016Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing...
Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5' UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.
Topics: 5' Untranslated Regions; Cell Cycle; Cell Cycle Proteins; F-Box Proteins; Fluorescence; Genes, Reporter; Genetic Techniques; Green Fluorescent Proteins; Humans; Luminescent Proteins; Optical Imaging; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA, Messenger; Ribosomes; Red Fluorescent Protein
PubMed: 27153498
DOI: 10.1016/j.cell.2016.04.034 -
Molecular Cell Aug 2022mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA...
mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA translation in a position-dependent manner. Although cytidine acetylation (ac4C) within protein-coding sequences stimulates translation, ac4C within 5' UTRs impacts protein synthesis at the level of initiation. 5' UTR acetylation promotes initiation at upstream sequences, competitively inhibiting annotated start codons. Acetylation further directly impedes initiation at optimal AUG contexts: ac4C within AUG-flanking Kozak sequences reduced initiation in base-resolved transcriptome-wide HeLa results and in vitro utilizing substrates with site-specific ac4C incorporation. Cryo-EM of mammalian 80S initiation complexes revealed that ac4C in the -1 position adjacent to an AUG start codon disrupts an interaction between C and hypermodified t6A at nucleotide 37 of the initiator tRNA. These findings demonstrate the impact of RNA modifications on nucleobase function at a molecular level and introduce mRNA acetylation as a factor regulating translation in a location-specific manner.
Topics: 5' Untranslated Regions; Animals; Codon, Initiator; Cytidine; Mammals; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA, Messenger
PubMed: 35679869
DOI: 10.1016/j.molcel.2022.05.016 -
Cell Nov 2022How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we...
How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.
Topics: Codon, Initiator; RNA, Messenger; 5' Untranslated Regions; Ribosomes; Saccharomyces cerevisiae; Adenosine Triphosphate; Peptide Chain Initiation, Translational; Protein Biosynthesis
PubMed: 36334590
DOI: 10.1016/j.cell.2022.10.005 -
Cell Aug 2022Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit...
Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.
Topics: Animals; Eukaryotic Initiation Factor-4G; Eukaryotic Initiation Factors; Poly(A)-Binding Proteins; Protein Biosynthesis; Purines; RNA, Messenger
PubMed: 35907403
DOI: 10.1016/j.cell.2022.06.037