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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 -
Cold Spring Harbor Perspectives in... Sep 2018This review summarizes our current understanding of translation in prokaryotes, focusing on the mechanistic and structural aspects of each phase of translation:... (Review)
Review
This review summarizes our current understanding of translation in prokaryotes, focusing on the mechanistic and structural aspects of each phase of translation: initiation, elongation, termination, and ribosome recycling. The assembly of the initiation complex provides multiple checkpoints for messenger RNA (mRNA) and start-site selection. Correct codon-anticodon interaction during the decoding phase of elongation results in major conformational changes of the small ribosomal subunit and shapes the reaction pathway of guanosine triphosphate (GTP) hydrolysis. The ribosome orchestrates proton transfer during peptide bond formation, but requires the help of elongation factor P (EF-P) when two or more consecutive Pro residues are to be incorporated. Understanding the choreography of transfer RNA (tRNA) and mRNA movements during translocation helps to place the available structures of translocation intermediates onto the time axis of the reaction pathway. The nascent protein begins to fold cotranslationally, in the constrained space of the polypeptide exit tunnel of the ribosome. When a stop codon is reached at the end of the coding sequence, the ribosome, assisted by termination factors, hydrolyzes the ester bond of the peptidyl-tRNA, thereby releasing the nascent protein. Following termination, the ribosome is dissociated into subunits and recycled into another round of initiation. At each step of translation, the ribosome undergoes dynamic fluctuations between different conformation states. The aim of this article is to show the link between ribosome structure, dynamics, and function.
Topics: Archaea; Bacteria; Gene Expression Regulation, Archaeal; Gene Expression Regulation, Bacterial; Prokaryotic Cells; Protein Biosynthesis
PubMed: 29661790
DOI: 10.1101/cshperspect.a032664 -
Cold Spring Harbor Perspectives in... Feb 2019Nonsense-mediated mRNA decay (NMD) is arguably the best-studied eukaryotic messenger RNA (mRNA) surveillance pathway, yet fundamental questions concerning the molecular... (Review)
Review
Nonsense-mediated mRNA decay (NMD) is arguably the best-studied eukaryotic messenger RNA (mRNA) surveillance pathway, yet fundamental questions concerning the molecular mechanism of target RNA selection remain unsolved. Besides degrading defective mRNAs harboring premature termination codons (PTCs), NMD also targets many mRNAs encoding functional full-length proteins. Thus, NMD impacts on a cell's transcriptome and is implicated in a range of biological processes that affect a broad spectrum of cellular homeostasis. Here, we focus on the steps involved in the recognition of NMD targets and the activation of NMD. We summarize the accumulating evidence that tightly links NMD to translation termination and we further discuss the recruitment and activation of the mRNA degradation machinery and the regulation of this complex series of events. Finally, we review emerging ideas concerning the mechanistic details of NMD activation and the potential role of NMD as a general surveyor of translation efficacy.
Topics: Codon, Nonsense; Eukaryota; Nonsense Mediated mRNA Decay; Peptide Chain Termination, Translational; Protein Biosynthesis; RNA, Messenger
PubMed: 29891560
DOI: 10.1101/cshperspect.a032862 -
Cold Spring Harbor Perspectives in... Oct 2018Termination of mRNA translation occurs when a stop codon enters the A site of the ribosome, and in eukaryotes is mediated by release factors eRF1 and eRF3, which form a... (Review)
Review
Termination of mRNA translation occurs when a stop codon enters the A site of the ribosome, and in eukaryotes is mediated by release factors eRF1 and eRF3, which form a ternary eRF1/eRF3-guanosine triphosphate (GTP) complex. eRF1 recognizes the stop codon, and after hydrolysis of GTP by eRF3, mediates release of the nascent peptide. The post-termination complex is then disassembled, enabling its constituents to participate in further rounds of translation. Ribosome recycling involves splitting of the 80S ribosome by the ATP-binding cassette protein ABCE1 to release the 60S subunit. Subsequent dissociation of deacylated transfer RNA (tRNA) and messenger RNA (mRNA) from the 40S subunit may be mediated by initiation factors (priming the 40S subunit for initiation), by ligatin (eIF2D) or by density-regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT1). These events may be subverted by suppression of termination (yielding carboxy-terminally extended read-through polypeptides) or by interruption of recycling, leading to reinitiation of translation near the stop codon.
Topics: Eukaryotic Cells; Peptide Termination Factors; Protein Biosynthesis; Protein Conformation; RNA, Messenger; Ribosomes
PubMed: 29735640
DOI: 10.1101/cshperspect.a032656 -
Trends in Cell Biology Jun 2020Eukaryotic cells must accurately monitor the integrity of the mitochondrial network to overcome environmental insults and respond to physiological cues. The... (Review)
Review
Eukaryotic cells must accurately monitor the integrity of the mitochondrial network to overcome environmental insults and respond to physiological cues. The mitochondrial unfolded protein response (UPR) is a mitochondrial-to-nuclear signaling pathway that maintains mitochondrial proteostasis, mediates signaling between tissues, and regulates organismal aging. Aberrant UPR signaling is associated with a wide spectrum of disorders, including congenital diseases as well as cancers and neurodegenerative diseases. Here, we review recent research into the mechanisms underlying UPR signaling in Caenorhabditis elegans and discuss emerging connections between the UPR signaling and a translational regulation program called the 'integrated stress response'. Further study of the UPR will potentially enable development of new therapeutic strategies for inherited metabolic disorders and diseases of aging.
Topics: Animals; Humans; Mitochondria; Protein Biosynthesis; Signal Transduction; Stress, Physiological; Unfolded Protein Response
PubMed: 32413314
DOI: 10.1016/j.tcb.2020.03.001 -
Cell Mar 2016Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three...
Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.
Topics: Animals; Humans; Protein Processing, Post-Translational; Proteins; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 27015313
DOI: 10.1016/j.cell.2016.03.003 -
Cell Jan 2023Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However,...
Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.
Topics: Carrier Proteins; Peptide Elongation Factors; Protein Biosynthesis; Ribosomal Proteins; Ribosomes; Ubiquitin-Protein Ligases; Ubiquitination; Humans; HeLa Cells; HEK293 Cells; RNA-Binding Proteins; Trans-Activators; Peptide Elongation Factor 1
PubMed: 36638793
DOI: 10.1016/j.cell.2022.12.025 -
Pharmacological Reviews Jan 2021G protein-coupled receptors (GPCRs) are a large family comprising >800 signaling receptors that regulate numerous cellular and physiologic responses. GPCRs have been... (Review)
Review
G protein-coupled receptors (GPCRs) are a large family comprising >800 signaling receptors that regulate numerous cellular and physiologic responses. GPCRs have been implicated in numerous diseases and represent the largest class of drug targets. Although advances in GPCR structure and pharmacology have improved drug discovery, the regulation of GPCR function by diverse post-translational modifications (PTMs) has received minimal attention. Over 200 PTMs are known to exist in mammalian cells, yet only a few have been reported for GPCRs. Early studies revealed phosphorylation as a major regulator of GPCR signaling, whereas later reports implicated a function for ubiquitination, glycosylation, and palmitoylation in GPCR biology. Although our knowledge of GPCR phosphorylation is extensive, our knowledge of the modifying enzymes, regulation, and function of other GPCR PTMs is limited. In this review we provide a comprehensive overview of GPCR post-translational modifications with a greater focus on new discoveries. We discuss the subcellular location and regulatory mechanisms that control post-translational modifications of GPCRs. The functional implications of newly discovered GPCR PTMs on receptor folding, biosynthesis, endocytic trafficking, dimerization, compartmentalized signaling, and biased signaling are also provided. Methods to detect and study GPCR PTMs as well as PTM crosstalk are further highlighted. Finally, we conclude with a discussion of the implications of GPCR PTMs in human disease and their importance for drug discovery. SIGNIFICANCE STATEMENT: Post-translational modification of G protein-coupled receptors (GPCRs) controls all aspects of receptor function; however, the detection and study of diverse types of GPCR modifications are limited. A thorough understanding of the role and mechanisms by which diverse post-translational modifications regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for improving and refining drug development for GPCRs.
Topics: Animals; Humans; Phosphorylation; Protein Processing, Post-Translational; Receptors, G-Protein-Coupled; Signal Transduction; Ubiquitination
PubMed: 33268549
DOI: 10.1124/pharmrev.120.000082 -
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 -
Nature Communications Apr 2023Although several ribosomal protein paralogs are expressed in a tissue-specific manner, how these proteins affect translation and why they are required only in certain...
Although several ribosomal protein paralogs are expressed in a tissue-specific manner, how these proteins affect translation and why they are required only in certain tissues have remained unclear. Here we show that RPL3L, a paralog of RPL3 specifically expressed in heart and skeletal muscle, influences translation elongation dynamics. Deficiency of RPL3L-containing ribosomes in RPL3L knockout male mice resulted in impaired cardiac contractility. Ribosome occupancy at mRNA codons was found to be altered in the RPL3L-deficient heart, and the changes were negatively correlated with those observed in myoblasts overexpressing RPL3L. RPL3L-containing ribosomes were less prone to collisions compared with RPL3-containing canonical ribosomes. Although the loss of RPL3L-containing ribosomes altered translation elongation dynamics for the entire transcriptome, its effects were most pronounced for transcripts related to cardiac muscle contraction and dilated cardiomyopathy, with the abundance of the encoded proteins being correspondingly decreased. Our results provide further insight into the mechanisms and physiological relevance of tissue-specific translational regulation.
Topics: Animals; Male; Mice; Muscle, Skeletal; Peptide Chain Elongation, Translational; Protein Biosynthesis; Ribosomal Proteins; Ribosomes; RNA, Messenger
PubMed: 37080962
DOI: 10.1038/s41467-023-37838-6