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High-throughput sequencing methods have led to the discovery of many non-coding RNAs, RNA modifications, and protein-RNA interactions. While the list keeps growing, the challenge of determining their functions remains. For our focus issue on RNA biology, we spoke with several researchers about their perspective on investigating the functions of RNA., Competing Interests: Declaration of interests L.Y. has an immediate family member on Molecular Cell’s advisory board. W.V.G. is a founder of Cloverleaf Bio and a member of its scientific advisory board. C.Y. has applied patents for a quantitative m(6)A detection method., (Copyright © 2024 Elsevier Inc. All rights reserved.)

All betacoronaviruses (β-CoVs) encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts host gene expression. Among the β-CoV family, MERS-CoV is the most distantly related member to SARS-CoV-2, and the mechanism for host translation inhibition by MERS-CoV Nsp1 remains controversial. Herein, we show that MERS-CoV Nsp1 directly interacts with the 40S ribosomal subunit. Using cryogenic electron microscopy (cryo-EM), we report a 2.6-Å structure of the MERS-CoV Nsp1 bound to the human 40S ribosomal subunit. The extensive interactions between C-terminal domain of MERS-CoV Nsp1 and the mRNA entry channel of the 40S ribosomal subunit are critical for its translation inhibition function. This mechanism of MERS-CoV Nsp1 is strikingly similar to SARS-CoV and SARS-CoV-2 Nsp1, despite modest sequence conservation. Our results reveal that the mechanism of host translation inhibition is conserved across β-CoVs and highlight a potential therapeutic target for the development of antivirals that broadly restrict β-CoVs., Competing Interests: Declaration of interests Y.X. serves on the advisory board of Cell Reports., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease., Competing Interests: Declaration of interests Sonya Neal, Fumiaki Ohtake, Ursula Jakob, Michael Lazarou, Wendy V. Gilbert, Sharon A. Tooze, and Kylie J. Walters declare no competing interests. Ana Maria Cuervo, Ramanujan S. Hegde, Zhijian J. Chen, James E. Haber, and F. Ulrich Hartl are members of the Molecular Cell advisory board., (Copyright © 2022. Published by Elsevier Inc.)

Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5' untranslated regions that confer 1,000-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we determined a functional role for most alternative 5' UTR isoforms expressed in yeast, revealed a general mode of increased translation via direct binding to a core translation factor, and identified numerous translational control elements including C-rich silencers that are sufficient to repress translation both in vitro and in vivo. DART enables systematic assessment of the translational regulatory potential of 5' UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing., Competing Interests: Declaration of interests G.W.Y. is the cofounder of, a member of the Board of Directors of, on the scientific advisory board of, an equity holder in, and a paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y.’s interests have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies. The authors declare no other competing financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Message-specific translational control is required for gametogenesis. In yeast, the RNA-binding protein Rim4 mediates translational repression of numerous mRNAs, including the B-type cyclin CLB3, which is essential for establishing the meiotic chromosome segregation pattern. Here, we show that Rim4 forms amyloid-like aggregates and that it is the amyloid-like form of Rim4 that is the active, translationally repressive form of the protein. Our data further show that Rim4 aggregation is a developmentally regulated process. Starvation induces the conversion of monomeric Rim4 into amyloid-like aggregates, thereby activating the protein to bring about repression of translation. At the onset of meiosis II, Rim4 aggregates are abruptly degraded allowing translation to commence. Although amyloids are best known for their role in the etiology of diseases such as Alzheimer's, Parkinson's, and diabetes by forming toxic protein aggregates, our findings show that cells can utilize amyloid-like protein aggregates to function as central regulators of gametogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cell survival in changing environments requires appropriate regulation of gene expression, including posttranscriptional regulatory mechanisms. From reporter gene studies in glucose-starved yeast, it was proposed that translationally silenced eukaryotic mRNAs accumulate in P bodies and can return to active translation. We present evidence contradicting the notion that reversible storage of nontranslating mRNAs is a widespread and general phenomenon. First, genome-wide measurements of mRNA abundance, translation, and ribosome occupancy after glucose withdrawal show that most mRNAs are depleted from the cell coincident with their depletion from polysomes. Second, only a limited subpopulation of translationally repressed transcripts, comprising fewer than 400 genes, can be reactivated for translation upon glucose readdition in the absence of new transcription. This highly selective posttranscriptional regulation could be a mechanism for cells to minimize the energetic costs of reversing gene-regulatory decisions in rapidly changing environments by transiently preserving a pool of transcripts whose translation is rate-limiting for growth., (Copyright © 2011 Elsevier Inc. All rights reserved.)