Similar Datasets
Project description:The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.
| S-EPMC6939389 | biostudies-literature
Project description:The Translation of Non-Canonical Open Reading Frames Controls Mucosal Immunity
| PRJNA494404 | ENA
Project description:A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.
| EGAS00001007426 | EGA
Project description:Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). Here, we exploit a systematic CRISPR-based screening strategy to identify hundreds of non-canonical CDSs that are essential for cellular growth and whose disruption elicit specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds that are presented by the HLA system. Interestingly, we find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same mRNA, thus revealing the diverse use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
2020-03-14 | GSE131650 | GEO
Project description:Protein-coding regions of the genome continue to expand beyond canonical annotations and remain incompletely understood. Despite recent reports demonstrating important cellular functions played by micropeptides from unannotated open reading frames (ORFs), there exists no systematic method to fully uncover the prevalence of functional ORFs and their encoded peptides. Here, using ribosome profiling and mass spectrometry based proteomics, we reveal pervasive translation of ORFs previously annotated as non-coding, including those encoded on long non-coding RNAs (lncRNA ORFs) and upstream regions of protein-coding messages (uORFs). Cross-validation of novel ORFs in six different cell lines via HLA peptidomics confirm their allotype-specific MHC receptor presentation and their engagement with the endogenous HLA processing machinery. We further developed systematic CRISPR-based screens to show that hundreds of these ORFs are essential for cellular growth and encode stable, functional peptides that play diverse roles in cells. By tagging with a split-GFP system, we show that many of the peptides have specific cellular localization patterns and form functional protein complexes. We uncovered uORFs encoding stable peptides that interact with the downstream protein encoded on the same mRNA, suggesting possible regulatory or feedback roles from the bicistronic message in addition to the previously assumed role of uORFs in translational repression. Our results uncover functional micropeptides encoded in the human genome with essential roles in diverse biological processes.
2020-03-06 | PXD014031 | Pride
Project description:In addition to canonical open reading frames (ORFs), thousands of translated small ORFs (containing less than 100 codons) have been identified in untranslated mRNA regions (UTRs) across eukaryotes. Small ORFs in 5' UTRs (upstream (u)ORFs) often repress translation of the canonical ORF within the same mRNA. However, the function of translated small ORFs in the 3' UTRs (downstream (d)ORFs) is unknown. Contrary to uORFs, we find that translation of dORFs enhances translation of their corresponding canonical ORFs. This translation stimulatory effect of dORFs depends on the number of dORFs, but not the length or peptide they encode. We propose that dORFs represent a new, strong, and universal translation regulatory mechanism in vertebrates.
| S-EPMC7459409 | biostudies-literature
Project description:Open reading frames (ORFs) are the genomic DNA sequences that have the potential to be translated. Genome annotation pipelines dismiss translation products of small ORFs (smORFs) of less than 100 codons (<300 nucleotides) as being unlikely to have a biological function. In this study, we systematically characterized smORFs in mouse B and T cells under different conditions and predicted a total of 5744 unique actively translated smORFs. We then extended our analysis to ORFs of 101-200 codons in length and predicted 945 of such longer translation products. Additionally, our results have suggested the existence of candidate secreted micropeptides. Furthermore, verifying their existence and identifying their functions will be essential and potentially lead to useful applications.
2021-04-28 | GSE154491 | GEO
Project description:Emerging evidence suggests that the cryptic translation beyond the annotated translatome may yield proteins with important function. However, the role and function mechanism of these cryptic ORFs in complex diseases such as cancer remain largely unknown. To fill this gap, we combined ribosome profiling and CRISPR/Cas9 screen to systematically identify the colorectal cancer (CRC) dependency on cryptic ORFs.
2023-08-06 | GSE184322 | GEO
Project description:Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
| S-EPMC7289059 | biostudies-literature
Project description:Macroautophagy/autophagy, a highly conserved catabolic pathway that maintains proper cellular homeostasis is stringently regulated by numerous autophagy-related (Atg) proteins. Many studies have investigated autophagy regulation at the transcriptional level; however, relatively little is known about translational control. Here, we report the upstream open reading frame (uORF)-mediated translational control of multiple Atg proteins in Saccharomyces cerevisiae and in human cells. The translation of several essential autophagy regulators in yeast, including Atg13, is suppressed by canonical uORFs under nutrient-rich conditions, and is activated during nitrogen-starvation conditions. We also found that the predicted human ATG4B and ATG12 non-canonical uORFs suppress downstream coding sequence translation. These results demonstrate that uORF-mediated translational control is a widely used mechanism among ATG genes from yeast to human and suggest a model for how some ATG genes bypass the general translational suppression that occurs under stress conditions to maintain a proper level of autophagy.Abbreviations: 5' UTR, 5' untranslated region; Atg, autophagy-related; CDS, coding sequence; Cvt, cytoplasm-to-vacuole targeting; HBSS, Hanks' balanced salt solution; PA, protein A; PE, phosphati-dylethanolamine; PIC, preinitiation complex; PtdIns3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative reverse transcription PCR; Ubl, ubiquitin-like; uORF, upstream open reading frame; WT, wild-type.
| S-EPMC9851245 | biostudies-literature