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The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs. We use interpretable statistical learning models to uncover GC content in the coding sequence as the major feature underlying RNA stabilization. This result corroborates GC content-related mRNA regulation detectable in other studies, including hundreds of ENCODE datasets and recent work focusing on mRNA dynamics in the cell cycle. We provide further evidence for mRNA stabilization by detailed analysis of RNA-seq profiles in hundreds of samples, including a Ddx3x conditional knockout mouse model exhibiting cell cycle and neurogenesis defects. Our study identifies a ubiquitous feature underlying mRNA regulation and highlights the importance of quantifying multiple steps of the gene expression cascade, where RNA abundance and protein production are often uncoupled.

Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

Mutations in the RNA helicase, DDX3X, are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet, the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is sensitive to Ddx3x dosage; complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y, compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms.

DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in the context of mRNA translation in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity- purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

Deep sequencing of ribosome footprints (ribosome profiling) maps and quantifies mRNA translation. Because ribosomes decode mRNA every 3 nt, the periodic property of ribosome footprints could be used to identify novel translated ORFs. However, due to the limited resolution of existing methods, the 3-nt periodicity is observed mostly in a global analysis, but not in individual transcripts. Here, we report a protocol applied to Arabidopsis that maps over 90% of the footprints to the main reading frame and thus offers super-resolution profiles for individual transcripts to precisely define translated regions. The resulting data not only support many annotated and predicted noncanonical translation events but also uncover small ORFs in annotated noncoding RNAs and pseudogenes. A substantial number of these unannotated ORFs are evolutionarily conserved, and some produce stable proteins. Thus, our study provides a valuable resource for plant genomics and an efficient optimization strategy for ribosome profiling in other organisms.

Deep sequencing of ribosome footprints (ribosome profiling) maps and quantifies mRNA translation. Because ribosomes decode mRNA every 3 nt, the periodic property of ribosome footprints could be used to identify novel translated ORFs. However, due to the limited resolution of existing methods, the 3-nt periodicity is observed mostly in a global analysis, but not in individual transcripts. Here, we report a protocol applied to Arabidopsis that maps over 90% of the footprints to the main reading frame and thus offers super-resolution profiles for individual transcripts to precisely define translated regions. The resulting data not only support many annotated and predicted noncanonical translation events but also uncover small ORFs in annotated noncoding RNAs and pseudogenes. A substantial number of these unannotated ORFs are evolutionarily conserved, and some produce stable proteins. Thus, our study provides a valuable resource for plant genomics and an efficient optimization strategy for ribosome profiling in other organisms. [ABSTRACT FROM AUTHOR]

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed., Competing Interests: Declaration of interests J.R.P. has received research honoraria from Novartis Biosciences and is a paid consultant for ProFound Therapeutics. J.G.A. is a paid consultant for Enara Bio and Moderna. J.L.A. is an advisor to Microneedle Solutions. T.F.M. is a consultant for and holds equity in Velia Therapeutics. J.S.W. is an advisor and holds equity in Velia Therapeutics. G.M. is co-founder and CSO of OHMX.bio. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer and PrognomIQ. N.T.I. hold equity in Velia Therapeutics and holds equity and serves as a scientific advisor to Tevard Biosciences. P.F. is a member of the scientific advisory board of Infinitopes. A.-R. C. is a member of the advisory board of ProFound Therapeutics.

The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs. We use interpretable statistical learning models to uncover GC content in the coding sequence as the major feature underlying RNA stabilization. This result corroborates GC content-related mRNA regulation detectable in other studies, including hundreds of ENCODE datasets and recent work focusing on mRNA dynamics in the cell cycle. We provide further evidence for mRNA stabilization by detailed analysis of RNA-seq profiles in hundreds of samples, including a Ddx3x conditional knockout mouse model exhibiting cell cycle and neurogenesis defects. Our study identifies a ubiquitous feature underlying mRNA regulation and highlights the importance of quantifying multiple steps of the gene expression cascade, where RNA abundance and protein production are often uncoupled.