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CRSPR/Cas9 gene knockout screen identifies colorectal cancer dependency on cryptic open reading frames

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184322 | GEO | 2023/08/06

REPOSITORIES: GEO

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CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.

Project description:Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

| S-EPMC10716047 | biostudies-literature

Identification of translated small open reading frames in lymphocytes

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

The Translation of Non-Canonical Open Reading Frames Controls Mucosal Immunity

Project description:We describe the identification of novel non-canonical ORFs in murine macrophages

2018-12-14 | GSE120762 | GEO

A spectral analysis approach to detects actively translated open reading frames in high resolution ribosome profiling data

Project description:RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions from Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of the three-nucleotide periodicity of Ribo-seq reads via spectral analysis methods. Examination of RNA fragments protected by ribosome

2015-11-12 | E-GEOD-73136 | biostudies-arrayexpress

Pervasive functional micropeptides in humans encoded by non-canonical open reading frames

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

A spectral analysis approach to detects actively translated open reading frames in high resolution ribosome profiling data

2015-11-12 | GSE73136 | GEO

Pervasive functional translation of noncanonical open reading frames

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

Accurate annotation of human protein-coding small open reading frames

Project description:Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs and provides an approach for identifying smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

2019-07-03 | GSE125218 | GEO

Conserved upstream open reading frames in higher plants.

Project description:BACKGROUND: Upstream open reading frames (uORFs) can down-regulate the translation of the main open reading frame (mORF) through two broad mechanisms: ribosomal stalling and reducing reinitiation efficiency. In distantly related plants, such as rice and Arabidopsis, it has been found that conserved uORFs are rare in these transcriptomes with approximately 100 loci. It is unclear how prevalent conserved uORFs are in closely related plants. RESULTS: We used a homology-based approach to identify conserved uORFs in five cereals (monocots) that could potentially regulate translation. Our approach used a modified reciprocal best hit method to identify putative orthologous sequences that were then analysed by a comparative R-nomics program called uORFSCAN to find conserved uORFs. CONCLUSION: This research identified new genes that may be controlled at the level of translation by conserved uORFs. We report that conserved uORFs are rare (<150 loci contain them) in cereal transcriptomes, are generally short (less than 100 nt), highly conserved (50% median amino acid sequence similarity), position independent in their 5'-UTRs, and their start codon context and the usage of rare codons for translation does not appear to be important.

| S-EPMC2527020 | biostudies-literature

Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq

Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits. Ribosomal profiling of large and small polysomal fractions in Drosophila S2 cells to assess translation of smORFs

2014-09-11 | E-GEOD-60384 | biostudies-arrayexpress

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