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Recent proteogenomic studies revealed extensive translation outside of annotated protein coding regions, such as non-coding RNAs and untranslated regions of mRNAs. This non-canonical translation is largely due to start codon plurality within the same RNA. This plurality is often due to the failure of some scanning ribosomes to recognize potential start codons leading to initiation downstream—a process termed leaky scanning. Codons other than AUG (non-AUG) are particularly leaky due to their inefficiency. Here we discuss our current understanding of non-AUG initiation. We argue for a near-ubiquitous role of non-AUG initiation in shaping the dynamic composition of mammalian proteomes.

Translational control shapes the proteome and is particularly important in regulating gene expression under stress. A key source of endothelial stress is treatment with tyrosine kinase inhibitors (TKIs), which lowers cancer mortality but increases cardiovascular mortality. Using a human induced pluripotent stem cell-derived endothelial cell (hiPSC-EC) model of sunitinib-induced vascular dysfunction combined with ribosome profiling, we assessed the role of translational control in hiPSC-ECs in response to stress. We identified staphylococcal nuclease and tudor domain-containing protein 1 (SND1) as a sunitinib-dependent translationally repressed gene. SND1 translational repression was mediated by the mTORC1/4E-BP1 pathway. SND1 inhibition led to endothelial dysfunction, whereas SND1 OE protected against sunitinib-induced endothelial dysfunction. Mechanistically, SND1 transcriptionally regulated UBE2N, an E2-conjugating enzyme that mediates K63-linked ubiquitination. UBE2N along with the E3 ligases RNF8 and RNF168 regulated the DNA damage repair response pathway to mitigate the deleterious effects of sunitinib. In silico analysis of FDA-approved drugs led to the identification of an ACE inhibitor, ramipril, that protected against sunitinib-induced vascular dysfunction in vitro and in vivo, all while preserving the efficacy of cancer therapy. Our study established a central role for translational control of SND1 in sunitinib-induced endothelial dysfunction that could potentially be therapeutically targeted to reduce sunitinib-induced vascular toxicity.

Ribosome profiling (Ribo-seq), a promising technology for exploring ribosome decoding rates, is characterized by the presence of infrequent high peaks in ribosome footprint density and by long alignment gaps. Here, to reduce the impact of data heterogeneity we introduce a simple normalization method, Ribo-seq Unit Step Transformation (RUST). RUST is robust and outperforms other normalization techniques in the presence of heterogeneous noise. We illustrate how RUST can be used for identifying mRNA sequence features that affect ribosome footprint densities globally. We show that a few parameters extracted with RUST are sufficient for predicting experimental densities with high accuracy. Importantly the application of RUST to 30 publicly available Ribo-seq data sets revealed a substantial variation in sequence determinants of ribosome footprint frequencies, questioning the reliability of Ribo-seq as an accurate representation of local ribosome densities without prior quality control. This emphasizes our incomplete understanding of how protocol parameters affect ribosome footprint densities. Ribosome profiling data can suffer from uneven coverage which hampers estimation of elongation rates. Connor et al. present an enhanced data smoothing method for Ribo-seq data and highlight significant variability in sequence determinants of ribosome density in publicly available data sets.

Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5′UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics.

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products. Proteins carry out essential tasks for living cells and genes contain the instructions to make proteins within their DNA. These instructions are copied to make a molecule of mRNA, and a molecular machine known as a ribosome then reads and translates the mRNA to build the protein. The first step in the translation process is called ‘initiation’ and requires a protein called eIF2 to work together with the ribosome. This step involves identifying an instruction called the start codon that marks the beginning of the mRNA's coding sequence. The section of an mRNA molecule before the start codon is not normally translated by the ribosome and is hence called the 5′ untranslated region. Building proteins requires energy and resources, and so it is carefully regulated. If a cell is stressed, such as by being exposed to harmful chemicals, it makes fewer proteins in order to conserve its resources. This down-regulation of protein production is achieved in part by the cell chemically modifying its eIF2 proteins to make them less able to initiate translation. However, stressed cells still continue to make more of certain proteins that help them to combat stress. The mRNA molecules for some of these proteins contain at least one other start codon in the 5′ untranslated region. The sequence that would be translated from such a start codon is known as an upstream open reading frame (or uORF for short)—and this feature is thought to help certain proteins to still be expressed despite low levels of active eIF2. Andreev, O'Connor et al. have now analysed which mRNAs are translated in human cells that have been treated with a chemical that induces stress and makes the eIF2 protein less able to initiate translation. To do so, a technique called ribosome profiling was used to identify all of the mRNA molecules bound to ribosomes shortly after treatment with this chemical. Overall translation of most mRNAs in stressed cells was reduced to a quarter of the normal level. However, Andreev, O'Connor et al. observed that the translation of a few mRNAs continued almost as normal, or even increased, after the chemical treatment. Notably, most of these mRNAs encoded regulatory proteins, which are not required in large amounts. With one exception, all of these resistant mRNAs contained uORFs. In unstressed cells, these uORFs were efficiently translated, while the same mRNA's coding sequences were translated less efficiently. Andreev, O'Connor et al. suggest that these two features could be used to identify mRNAs that are still translated into working proteins when cells are stressed. Further work is now needed to explore the mechanisms by which translation of these uORFs allows mRNAs to resist the stress.

The synthesis of most proteins begins at AUG codons, yet a small number of non-AUG initiated proteoforms are also known. Here we analyse a large number of publicly available Ribo-seq datasets to identify novel, previously uncharacterised non-AUG proteoforms using Trips-Viz implementation of a novel algorithm for detecting translated ORFs. In parallel we analyse genomic alignment of 120 mammals to identify evidence of protein coding evolution in sequences encoding potential extensions. Unexpectedly we find that the number of non-AUG proteoforms identified with ribosome profiling data greatly exceeds those with strong phylogenetic support suggesting their recent evolution. Our study argues that the protein coding potential of human genome greatly exceeds that detectable through comparative genomics and exposes the existence of multiple proteins encoded by the same genomic loci. Analysis of a large number of Ribo-seq datasets and genomic alignments led to detection of novel non-AUG proteoforms. Unexpectedly the number of non-AUG proteoforms identified with Ribo-seq greatly exceeds those with strong phylogenetic support.

Ribosome profiling is a powerful technique used to study gene expression on a transcriptome-wide scale. It involves sequencing of mRNA fragments protected by ribosomes from ribonuclease digestion. The initial steps commonly involve cell lysis followed by centrifugation and ribonuclease digestion. We find that centrifugation depletes 329 translated mRNAs in HEK293T cells. Many of these mRNAs encode cytoskeleton proteins. This suggests that the expression of a subset of mRNAs may be significantly underestimated in most ribosome profiling experiments. We show that omitting the centrifugation step after cell lysis can resolve this issue.

Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death. Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames. These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.

The study of nitrogen assimilation in yeast is of interest from genetic, evolutionary, and biotechnological perspectives. Over the course of evolution, yeasts have developed sophisticated control mechanisms to regulate nitrogen metabolism, with domesticated lineages sometimes displaying particular specialisation. The focus of this study was on assimilation of asparagine, which is a significant nutritional source for some alcoholic fermentations. We were particularly interested in ASP3, which encodes a periplasmic asparaginase and that was proposed to have been acquired relatively recently in S. cerevisiae by horizontal gene transfer. We examined 1680 S. cerevisiae genome assemblies to evaluate the distribution and evolutionary trajectory of ASP3. Our findings suggest an alternative hypothesis that ASP3 is an ancient Saccharomyces gene that has generally been lost over the course of evolution but has been retained in certain fermentative environments. As asparagine is the major nitrogen source in apple juice, we explored whether the presence of ASP3 would confer a growth advantage. Interestingly, we found that although ASP3 enhances growth when asparagine is the sole nitrogen source, the same effect is not seen in apple juice. These data indicate that growth in pure culture may not reflect the original selective environment for ASP3+ strains and highlight the role that complex regulation may play in optimising nitrogen assimilation in yeasts.