Search

Ded1 and Dbp1 are paralogous conserved DEAD-box ATPases involved in translation initiation in yeast. In long-term starvation states, Dbp1 expression increases and Ded1 decreases, whereas in cycling mitotic cells, Dbp1 is absent. Inserting DBP1 in place of DED1 cannot replace Ded1 function in supporting mitotic translation, partly due to inefficient translation of the DBP1 coding region. Global translation measurements, activity of mRNA-tethered proteins, and growth assays show that-even at matched protein levels-Ded1 is better than Dbp1 at activating translation, especially for mRNAs with structured 5' leaders. Heat-stressed cells normally downregulate translation of structured housekeeping transcripts and halt growth, but neither occurs in Dbp1-expressing cells. This failure to halt growth in response to heat is not based on deficient stress granule formation or failure to reduce bulk translation. Rather, it depends on heat-triggered loss of Ded1 function mediated by an 11-amino-acid interval within its intrinsically disordered C terminus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the earliest contact between mRNAs and the translation machinery, offering a key regulatory nexus. We acutely depleted this essential protein and found surprisingly modest effects on cell growth and recovery of protein synthesis. Paradoxically, impaired protein biosynthesis upregulated genes involved in the catabolism of aromatic amino acids simultaneously with the induction of the amino acid biosynthetic regulon driven by the integrated stress response factor GCN4. We further identified the translational control of Pho85 cyclin 5 (PCL5), a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This regulation depended in part on a uniquely long poly(A) tract in the PCL5 5' UTR and poly(A) binding protein. Collectively, these results highlight how eIF4E connects protein synthesis to metabolic gene regulation, uncovering mechanisms controlling translation during environmental challenges., Competing Interests: Declaration of interests N.T.I. declares equity in Tevard Biosciences and Velia Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In the first of many thematic issues marking the 30 th anniversary of Cell Chemical Biology, we highlight the contribution of chemical biology to RNA biology in a special issue on RNA modulation. We asked several leaders in the field to share their opinions on the current challenges and opportunities in RNA biology., Competing Interests: Declaration of interests Shalini Oberdoerffer declares no competing interests. Ling-Ling Chen is a member of the Cell Chemical Biology advisory board. Nicholas T. Ingolia holds equity in Tevard Biosciences and is a member of the scientific advisory board. Nicholas T. Ingolia holds equity in Velia Therapeutics. Megan L. Insco is an inventor on patent application no. PCT/US2022/037839, filed on 07-21-2022 by the Children’s Medical Center Corporation and titled “Selective nuclear RNA degradation as a therapeutic mechanism.” Jin Billy Li is a co-founder and advisor of AIRNA and an inventor of patents related to the RNA base-editing technology. Kevin M. Weeks is an advisor to and holds equity in Ribometrix., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus., Competing Interests: Declaration of interests B.A.G. is a member of the Vaccine Advisory Board for Celsion Corporation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In Cell Chemical Biology, Naineni et al. (2021) report the structure of a synthetic analog of the translation inhibitor pateamine A bound to initiation factor eIF4A and RNA. The drug stacks with RNA bases in the same pocket where unrelated rocaglate compounds bind, highlighting a druggable site for DEAD-box proteins., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). This mutation results in dopaminergic neurodegeneration via dysregulated protein translation, although how alterations in protein synthesis contribute to neurodegeneration in human neurons is not known. Here we define the translational landscape in LRRK2-mutant dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) via ribosome profiling. We found that mRNAs that have complex secondary structure in the 5' untranslated region (UTR) are translated more efficiently in G2019S LRRK2 neurons. This leads to the enhanced translation of multiple genes involved in Ca 2+ regulation and to increased Ca 2+ influx and elevated intracellular Ca 2+ levels, a major contributor to PD pathogenesis. This study reveals a link between dysregulated translation control and Ca 2+ homeostasis in G2019S LRRK2 human dopamine neurons, which potentially contributes to the progressive and selective dopaminergic neurotoxicity in PD., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diverse ribonucleoprotein complexes control mRNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS), obtained precisely in parallel, provides new insights into the organization of translation initiation complexes on active mRNAs and unanticipated complexity in stress granule composition. Our novel technique allows a powerful and general approach to explore the spatial environment of macromolecules., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Once inactive ribosomes are accounted for, ribosome footprint profiling data and pulse-labeled proteomics provide similar, accurate measurements of protein synthesis., (Copyright © 2017. Published by Elsevier Inc.)

Protein synthesis continues in platelets and maturing reticulocytes, although these blood cells lack nuclei and do not make new mRNA or ribosomes. Here, we analyze translation in primary human cells from anucleate lineages by ribosome profiling and uncover a dramatic accumulation of post-termination unrecycled ribosomes in the 3' UTRs of mRNAs. We demonstrate that these ribosomes accumulate as a result of the natural loss of the ribosome recycling factor ABCE1 during terminal differentiation. Induction of the ribosome rescue factors PELO and HBS1L is required to support protein synthesis when ABCE1 levels fall and for hemoglobin production during blood cell development. Our observations suggest that this distinctive loss of ABCE1 in anucleate blood lineages could sensitize them to defects in ribosome homeostasis, perhaps explaining in part why genetic defects in the fundamental process of ribosome production ("ribosomopathies") often affect hematopoiesis specifically., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribosome profiling has emerged as a technique for measuring translation comprehensively and quantitatively by deep sequencing of ribosome-protected mRNA fragments. By identifying the precise positions of ribosomes, footprinting experiments have unveiled key insights into the composition and regulation of the expressed proteome, including delineating potentially functional micropeptides, revealing pervasive translation on cytosolic RNAs, and identifying differences in elongation rates driven by codon usage or other factors. This Primer looks at important experimental and analytical concerns for executing ribosome profiling experiments and surveys recent examples where the approach was developed to explore protein biogenesis and homeostasis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A fundamental goal of genomics is to identify the complete set of expressed proteins. Automated annotation strategies rely on assumptions about protein-coding sequences (CDSs), e.g., they are conserved, do not overlap, and exceed a minimum length. However, an increasing number of newly discovered proteins violate these rules. Here we present an experimental and analytical framework, based on ribosome profiling and linear regression, for systematic identification and quantification of translation. Application of this approach to lipopolysaccharide-stimulated mouse dendritic cells and HCMV-infected human fibroblasts identifies thousands of novel CDSs, including micropeptides and variants of known proteins, that bear the hallmarks of canonical translation and exhibit translation levels and dynamics comparable to that of annotated CDSs. Remarkably, many translation events are identified in both mouse and human cells even when the peptide sequence is not conserved. Our work thus reveals an unexpected complexity to mammalian translation suited to provide both conserved regulatory or protein-based functions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aging is associated with the decline of protein, cell, and organ function. Here, we use an integrated approach to characterize gene expression, bulk translation, and cell biology in the brains and livers of young and old rats. We identify 468 differences in protein abundance between young and old animals. The majority are a consequence of altered translation output, that is, the combined effect of changes in transcript abundance and translation efficiency. In addition, we identify 130 proteins whose overall abundance remains unchanged but whose sub-cellular localization, phosphorylation state, or splice-form varies. While some protein-level differences appear to be a generic property of the rats' chronological age, the majority are specific to one organ. These may be a consequence of the organ's physiology or the chronological age of the cells within the tissue. Taken together, our study provides an initial view of the proteome at the molecular, sub-cellular, and organ level in young and old rats., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (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: copurification 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 the 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 at understanding how cells manage and exploit its consequences., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full-length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA and suggests a widespread role for polycistronic mRNAs in the innate immune system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell's life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. PAPERCLIP:, (Copyright © 2013 Elsevier Inc. All rights reserved.)

Large noncoding RNAs are emerging as an important component in cellular regulation. Considerable evidence indicates that these transcripts act directly as functional RNAs rather than through an encoded protein product. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. Here, we show that classical noncoding RNAs and 5' UTRs show the same ribosome occupancy as lincRNAs, demonstrating that ribosome occupancy alone is not sufficient to classify transcripts as coding or noncoding. Instead, we define a metric based on the known property of translation whereby translating ribosomes are released upon encountering a bona fide stop codon. We show that this metric accurately discriminates between protein-coding transcripts and all classes of known noncoding transcripts, including lincRNAs. Taken together, these results argue that the large majority of lincRNAs do not function through encoded proteins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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 unannotated 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 class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Bistability in genetic networks allows cells to remember past events and to make discrete decisions in response to graded signals. Bistable behavior can result from positive feedback, but feedback loops can have other roles in signal transduction as well., Results: We introduced positive feedback into the budding-yeast pheromone response to convert it into a bistable system. In the presence of feedback, transient induction with high pheromone levels caused persistent pathway activation, whereas at lower levels a fraction of cells became persistently active but the rest inactivated completely. We also generated mutations that quantitatively tuned the basal and induced expression levels of the feedback promoter and showed that they qualitatively changed the behavior of the system. Finally, we developed a simple stochastic model of our positive-feedback system and showed the agreement between our simulations and experimental results., Conclusions: The positive-feedback loop can display several different behaviors, including bistability, and can switch between them as a result of simple mutations.

We discuss the impact of mathematical modeling on our understanding of the cell cycle. Although existing, detailed models confirm that the known interactions in the cell cycle can produce oscillations and predict behaviors such as hysteresis, they contain many parameters and are poorly constrained by data which are almost all qualitative. Questions about the basic architecture of the oscillator may be more amenable to modeling approaches that ignore molecular details. These include asking how the various elaborations of the basic oscillator affect the robustness of the system and how cells monitor their size and use this information to control the cell cycle.