PMCDS000ABG - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001011193 | EGA |
REPOSITORIES: EGA
Similar Datasets
Project description:This study aims to investigate the dysregulation of RNA translation and identify functional non-canonical open reading frames (ORFs) as potential targets for medulloblastoma treatment. The study involves ribosome profiling and RNAseq of medulloblastoma tissues and cell lines to observe the translation of non-canonical ORFs. Multiple CRISPR-Cas9 screens will be used to identify functional non-canonical ORFs implicated in medulloblastoma cell survival.
| EGAD00001011192 | EGA
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 using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins 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. A microprotein encoded by one of these ORFs, 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 studies seeking to define new cancer targets.
2024-01-03 | PXD046091 | Pride
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:Open reading frame (ORF) boundaries in bacterial genomes have largely been drawn by gene prediction algorithms. However, these algorithms often fail to predict ORFs with non-canonical features, including those that are short, overlapping, or lack 5’ UTRs. Recent developments in genome-scale mapping of translation have facilitated the empirical identification of open reading frames (ORFs). Here, we use ribosome profiling approaches to map initiating and elongating ribosomes in Mycobacterium tuberculosis. Thus, we identify over 1,000 novel ORFs, revealing that much of the M. tuberculosis genome encodes proteins in overlapping reading frames, and/or on both strands. Most of the novel ORFs are short (sORFs), impeding their identification by traditional methods. The strong codon bias that characterizes annotated mycobacterial ORFs is not evident in the aggregate novel sORFs, and hence most are unlikely to encode functional proteins. Thus, our data suggest that bacterial transcriptomes are subject to pervasive translation that occurs as a result of the relatively low specificity requirements of initiating ribosomes. We speculate that the inefficiency of expressing spurious sORFs may be offset by positive contributions to M. tuberculosis biology through cis and trans regulatory activities of a small subset.
2019-06-20 | E-MTAB-8039 | biostudies-arrayexpress
Project description:The Translation of Non-Canonical Open Reading Frames Controls Mucosal Immunity
| PRJNA494404 | ENA
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:Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. However, curation and stringent validation further reduces this to 10 reliable identifications. The fragmentation of these 10 peptides was further validated by comparison with the fragmentation of synthetic peptides.
2022-08-02 | PXD030285 | panorama
Project description:Pervasive functional translation of noncanonical open reading frames
| PRJNA544411 | ENA
Project description:Translation initiation generally occurs at AUG codons in eukaryotes although it has been shown that non-AUG or non-canonical translation initiation can also occur. However, the evidence for non-canonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling studies. Here, using a strategy specifically designed to enrich N-termini of proteins, we demonstrate that many human proteins are translated at non-canonical TISs. The large majority of TISs that mapped to 5’ untranslated regions were non-canonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). There has been little controversy on whether the corresponding amino acid to the start codon is incorporated at TIS or methionine is still incorporated. Notably, methionine was incorporated at almost all non-canonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by ribosome profiling data. The sequence orthology analysis and relative translation frequency analysis of non-canonical TISs over canonical ones suggests that those non-canonical TISs are not leaky but they can have certain biological functions. Overall, this study provides evidence of protein translation initiation at non-canonical TISs and indicates that further studies are required for elucidation of functional implications of such non-canonical translation initiation.
2017-11-23 | PXD006633 | Pride
Project description:Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins and might be functional, while other studies failed to find evidence for such types of non-canonical translation events. In this study we tried to detect (and characterize) proteins originating from these non-translated regions (NTRs). We attempted to discover translation products from non-coding regions at large scale by reducing the overall sample complexity (by enriching cytosolic N-terminal peptides) and combined it with an extend search space (combining UniProt proteins, UniProt isoforms and publicly available Ribo-seq data). Reasoning that this strategy would increase the likelihood of identifying proteins from NTRs. Further, we introduced rigorous data analysis and results curation workflows. This stringent filtering approach was found essential to retain confident translational evidence at the peptide level for NTRs. We show that, theoretically, our strategy facilitates the detection of translation events of transcripts from NTRs, but experimentally we find that less than 1% of all identified peptides might originate from such translation events. However, one NTR protein was further characterized by Virotrap based interaction analysis. This resulted in several potential interaction partners associated with membranes and vesicle transport. Showing that the non-translated regions that do result in proteins might be functional.
2022-07-12 | PXD030216 | Pride