Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

Cell death or survival: Insights into the role of mRNA translational control.

Cellular stress is an intrinsic part of cell physiology that underlines cell survival or death. The ability of mammalian cells to regulate global protein synthesis (aka translational control) represents a critical, yet underappreciated, layer of regulation during the stress response. Various cellular stress response pathways monitor conditions of cell growth and subsequently reshape the cellular translatome to optimize translational outputs. On the molecular level, such translational reprogramming involves an intricate network of interactions between translation machinery, RNA-binding proteins, mRNAs, and non-protein coding RNAs. In this review, we will discuss molecular mechanisms, signaling pathways, and targets of translational control that contribute to cellular adaptation to stress and to cell survival or death.

Editing and genome-wide analysis upstream open reading frames contributes to enhancing salt tolerance in tomato.

The salinization of soil constitutes a substantial hindrance to the advancement of sustainable agriculture. Our research seeks to elucidate the role of a Rab GTPase-activating protein (RabGAP) family member, SlRabGAP22, in salt tolerance and its translational regulation under salt stress in tomatoes, employing gene-editing techniques and ribosome profiling methodologies. Findings demonstrate that SlRabGAP22 acts as a positive regulator of tomato salt tolerance, with four predicted upstream open reading frames (uORFs) classified into three categories. Functional uORFs were found to be negative regulation. Editing these uORFs along with altering their classifications and characteristics mitigated the inhibitory effects on primary ORFs and fine-tuned gene expression. Enhanced tomato salt tolerance was attributed to improved scavenging of reactive oxygen species, reduced toxicity Na+, and diminished osmotic stress effects. Furthermore, we conducted genome-wide analysis of ORFs to lay the foundation for further research on uORFs in tomatoes. In summary, our findings offer novel perspectives and important data for the enhancement of genetic traits via uORF-based strategies and translational regulation against the backdrop of salt stress.

Widespread polycistronic gene expression in green algae.

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: Chlamydomonas reinhardtii and Chromochloris zofingiensis Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.

Androgen Insensitivity Syndrome DUE to Non-Coding Variation in the Androgen Receptor Gene: Review of the Literature and Case Report of a Patient with Mosaic c.-547C>T Variant.

Sexual development (SD) is a complex process with strict spatiotemporal regulation of gene expression. Despite advancements in molecular diagnostics, disorders of sexual development (DSD) have a diagnostic rate of ~50%. Androgen insensitivity syndrome (AIS) represents the most common form of 46,XY DSD, with a spectrum of defects in androgen action. Considering the importance of very strict regulation of the SD, it is reasonable to assume that the genetic cause for proportion of the DSD lies in the non-coding part of the genome that regulates proper gene functioning. Here we present a patient with partial AIS (PAIS) due to a mosaic de novo c.-547C>T pathogenic variant in the 5'UTR of androgen receptor (AR) gene. The same mutation was previously described as inherited, in two unrelated patients with complete AIS (CAIS). Thus, our case further confirms the previous findings that variable gene expressivity could be attributed to mosaicism. Mutations in 5'UTR could create new upstream open reading frames (uORFs) or could disrupt the existing one. A recent systematic genome-wide study identified AR as a member of a subset of genes where modifications of uORFs represents an important disease mechanism. Only a small number of studies are reporting non-coding mutations in the AR gene and our case emphasizes the importance of molecular testing of the entire AR locus in AIS patients. The introduction of new methods for comprehensive molecular testing in routine genetic diagnosis, accompanied with new tools for in sillico analysis could improve the genetic diagnosis of AIS, and DSD in general.

Inhibition of eIF6 Activity Reduces Hepatocellular Carcinoma Growth: An In Vivo and In Vitro Study.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.

The Role and Therapeutic Potential of the Integrated Stress Response in Amyotrophic Lateral Sclerosis.

In amyotrophic lateral sclerosis (ALS) patients, loss of cellular homeostasis within cortical and spinal cord motor neurons triggers the activation of the integrated stress response (ISR), an intracellular signaling pathway that remodels translation and promotes a gene expression program aimed at coping with stress. Beyond its neuroprotective role, under regimes of chronic or excessive stress, ISR can also promote cell/neuronal death. Given the two-edged sword nature of ISR, many experimental attempts have tried to establish the therapeutic potential of ISR enhancement or inhibition in ALS. This review discusses the complex interplay between ISR and disease progression in different models of ALS, as well as the opportunities and limitations of ISR modulation in the hard quest to find an effective therapy for ALS.

ORFik: a comprehensive R toolkit for the analysis of translation.

BACKGROUND: With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. RESULTS: Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5'UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5' UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. CONCLUSION: In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment. AVAILABILITY: http://bioconductor.org/packages/ORFik .

Some like it translated: small ORFs in the 5'UTR.

The 5' untranslated region (5'UTR) is critical in determining post-transcriptional control, which is partly mediated by short upstream open reading frames (uORFs) present in half of mammalian transcripts. uORFs are generally considered to provide functionally important repression of the main-ORF by engaging initiating ribosomes, but under specific environmental conditions such as cellular stress, uORFs can become essential to activate the translation of the main coding sequence. In addition, a growing number of uORF-encoded bioactive microproteins have been described, which have the potential to significantly increase cellular protein diversity. Here we review the diverse cellular contexts in which uORFs play a critical role and discuss the molecular mechanisms underlying their function and regulation. The progress over the last decades in dissecting uORF function suggests that the 5'UTR remains an exciting frontier towards understanding how the cellular proteome is shaped in health and disease.

Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops.

Global population growth and climate change are posing increasing challenges to the production of a stable crop supply using current agricultural practices. The generation of genetically modified (GM) crops has contributed to improving crop stress tolerance and productivity; however, many regulations are still in place that limit their commercialization. Recently, alternative biotechnology-based strategies, such as gene-edited (GE) crops, have been in the spotlight. Gene-editing technology, based on the clustered regularly interspaced short palindromic repeats (CRISPR) platform, has emerged as a revolutionary tool for targeted gene mutation, and has received attention as a game changer in the global biotechnology market. Here, we briefly introduce the concept of upstream open reading frames (uORFs) editing, which allows for control of the translation of downstream ORFs, and outline the potential for enhancing target gene expression by mutating uORFs. We discuss the current status of developing stress-tolerant crops, and discuss uORF targets associated with salt stress-responsive genes in rice that have already been verified by transgenic research. Finally, we overview the strategy for developing GE crops using uORF editing via the CRISPR-Cas9 system. A case is therefore made that the mutation of uORFs represents an efficient method for developing GE crops and an expansion of the scope of application of genome editing technology.

New Peptides Under the s(ORF)ace of the Genome.

Hundreds, perhaps thousands of previously unidentified functional small peptides could exist in most genomes, but these sequences have been generally overlooked. The discovery of genes encoding small peptides with important functions in different organisms has ignited the interest in these sequences and led to an increasing amount of effort towards their identification. Here, we review the advances, both computational and biochemical, that are leading the way in the discovery of putatively functional small open reading frame genes (smORFs), as well as the functional studies that have been carried out as a consequence of these searches. The evidence suggests that smORFs form a substantial part of our genomes, and that their encoded peptides could have a variety of important cellular functions.

Upstream ORFs are prevalent translational repressors in vertebrates.

Regulation of gene expression is fundamental in establishing cellular diversity and a target of natural selection. Untranslated mRNA regions (UTRs) are key mediators of post-transcriptional regulation. Previous studies have predicted thousands of ORFs in 5'UTRs, the vast majority of which have unknown function. Here, we present a systematic analysis of the translation and function of upstream open reading frames (uORFs) across vertebrates. Using high-resolution ribosome footprinting, we find that (i)uORFs are prevalent within vertebrate transcriptomes, (ii) the majority show signatures of active translation, and (iii)uORFs act as potent regulators of translation and RNA levels, with a similar magnitude to miRNAs. Reporter experiments reveal clear repression of downstream translation by uORFs/oORFs. uORF number, intercistronic distance, overlap with the CDS, and initiation context most strongly influence translation. Evolution has targeted these features to favor uORFs amenable to regulation over constitutively repressive uORFs/oORFs. Finally, we observe that the regulatory potential of uORFs on individual genes is conserved across species. These results provide insight into the regulatory code within mRNA leader sequences and their capacity to modulate translation across vertebrates.

GOLT1A-KISS1 fusion is associated with metastasis in adenoid cystic carcinomas.

Genetic alterations can drive carcinogenesis. Numerous studies have shown that gene fusion is associated with cancer progression and could provide valuable biomarkers for clinical diagnosis or targets for cancer therapy. Adenoid cystic carcinoma (ACC) is a rare form of adenocarcinoma, characterized by frequent local recurrence and high rates of distant metastasis, ultimately resulting in low survival rates. Owing to the lack of effective therapeutic targets and limited biomarkers for diagnosis, a deeper understanding of the molecular basis of ACC is urgently needed. Here, we show that gene fusion is associated with ACC metastasis. We identified a metastasis suppressor KISS1 fused with a close-by gene, GOLT1A, in highly metastatic ACC cell lines and human specimens. Such fusion blocks KISS1 translation, but not transcription, by introducing 5' upstream open reading frames (uORFs) in the GOLT1A-KISS1 fusion transcript. Deletion of these uORFs rescued KISS1 expression and reduced invasion and migration of metastatic ACC cells. We also detected GOLT1A-KISS1 fusion transcripts in other types of highly metastatic cancer cell lines. Taken together, our results highlight the significance of this novel GOLT1A-KISS1 gene fusion in tumor metastasis and provide a valuable biomarker for clinical diagnosis and future therapeutic targeting of ACC.

An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression.

Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.

uORFs: Important Cis-Regulatory Elements in Plants.

Gene expression is regulated at many levels, including mRNA transcription, translation, and post-translational modification. Compared with transcriptional regulation, mRNA translational control is a more critical step in gene expression and allows for more rapid changes of encoded protein concentrations in cells. Translation is highly regulated by complex interactions between cis-acting elements and trans-acting factors. Initiation is not only the first phase of translation, but also the core of translational regulation, because it limits the rate of protein synthesis. As potent cis-regulatory elements in eukaryotic mRNAs, upstream open reading frames (uORFs) generally inhibit the translation initiation of downstream major ORFs (mORFs) through ribosome stalling. During the past few years, with the development of RNA-seq and ribosome profiling, functional uORFs have been identified and characterized in many organisms. Here, we review uORF identification, uORF classification, and uORF-mediated translation initiation. More importantly, we summarize the translational regulation of uORFs in plant metabolic pathways, morphogenesis, disease resistance, and nutrient absorption, which open up an avenue for precisely modulating the plant growth and development, as well as environmental adaption. Additionally, we also discuss prospective applications of uORFs in plant breeding.

Seq-ing answers: uncovering the unexpected in global gene regulation.

The development of techniques for measuring gene expression globally has greatly expanded our understanding of gene regulatory mechanisms in depth and scale. We can now quantify every intermediate and transition in the canonical pathway of gene expression-from DNA to mRNA to protein-genome-wide. Employing such measurements in parallel can produce rich datasets, but extracting the most information requires careful experimental design and analysis. Here, we argue for the value of genome-wide studies that measure multiple outputs of gene expression over many timepoints during the course of a natural developmental process. We discuss our findings from a highly parallel gene expression dataset of meiotic differentiation, and those of others, to illustrate how leveraging these features can provide new and surprising insight into fundamental mechanisms of gene regulation.

Eukaryotic Initiation Factor 5B (eIF5B) Cooperates with eIF1A and eIF5 to Facilitate uORF2-Mediated Repression of ATF4 Translation.

A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B-which can substitute for eIF2 in delivering Met-tRNAi-leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.

Kinases of eIF2a Switch Translation of mRNA Subset during Neuronal Plasticity.

Compared to other types of cells, neurons express the largest number of diverse mRNAs, including neuron-specific ones. This mRNA diversity is required for neuronal function, memory storage, maintenance and retrieval. Regulation of translation in neurons is very complicated and involves various proteins. Some proteins, implementing translational control in other cell types, are used by neurons for synaptic plasticity. In this review, we discuss the neuron-specific activity of four kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), general control nonderepressible 2 kinase (GCN2), and heme-reguated eIF2α kinase (HRI), the substrate for which is α-subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α is necessary for the cell during stress conditions, such as lack of amino acids, energy stress or viral infection. We propose that, during memory formation, neurons use some mechanisms similar to those involved in the cellular stress. The four eIF2α kinases regulate translation of certain mRNAs containing upstream open reading frames (uORFs). These mRNAs encode proteins involved in the processes of long-term potentiation (LTP) or long-term depression (LTD). The review examines some neuronal proteins for which translation regulation by eIF2 was suggested and checked experimentally. Of such proteins, we pay close attention to protein kinase Mζ, which is involved in memory storage and regulated at the translational level.

Ribosome profiling reveals dynamic translational landscape in maize seedlings under drought stress.

Plants can respond to environmental changes with various mechanisms occurred at transcriptional and translational levels. Thus far, there have been relatively extensive understandings of stress responses of plants on transcriptional level, while little information is known about that on translational level. To uncover the landscape of translation in plants in response to drought stress, we performed the recently developed ribosome profiling assay with maize seedlings growing under normal and drought conditions. Comparative analysis of the ribosome profiling data and the RNA-seq data showed that the fold changes of gene expression at transcriptional level were moderately correlated with that of translational level globally (R(2) = 0.69). However, less than half of the responsive genes were shared by transcription and translation under drought condition, suggesting that drought stress can introduce transcriptional and translational responses independently. We found that the translational efficiencies of 931 genes were changed significantly in response to drought stress. Further analysis revealed that the translational efficiencies of genes were highly influenced by their sequence features including GC content, length of coding sequences and normalized minimal free energy. In addition, we detected potential translation of 3063 upstream open reading frames (uORFs) on 2558 genes and these uORFs may affect the translational efficiency of downstream main open reading frames (ORFs). Our study indicates that plant can respond to drought stress with highly dynamic translational mechanism, that acting synergistically with that of transcription.