Differential gene expression analyses after noncanonical peptide overexpression
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE216093 | GEO | 2024/02/01
REPOSITORIES: GEO
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Project description:Mass spectrometry peptidomics coupled with RNA deep sequencing has revealed a large number of translated eukaryotic short open reading frames that produce genomically encoded peptides. The human LOC550643 gene, previously annotated as non-coding, encodes such a peptide (referred to as NoBody). NoBody peptide binds to EDC4 (enhancer of decapping 4) site-specifically, enriches complexes of proteins involved in 5'-3' mRNA degradation, and localizes to P-bodies, which are cellular RNA-protein granules associated with RNA degradation. Furthermore, modulating NoBody expression levels inversely regulates P-body numbers. This peptide has the potential to reveal new insights into the P-body structure-function relationship and cellular regulation of mRNA degradation. We used siRNA-mediated gene silencing coupled with biological pathway analysis to determine the cellular functions of NoBody (LOC550643), and whether it has similar cellular functions to related (Dcp2) and unrelated (DcpS) mRNA degradation proteins.
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Project description:Tumor epitopes – peptides that are presented on surface-bound MHC I proteins - provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples. These included primary normal and chronic lymphocytic leukemia (CLL) B cells, patient-derived primary glioblastoma (GBM) and melanoma cell cultures, primary healthy melanocytes, as well as established colon carcinoma and melanoma cell lines. These also included B721.221 cells, the parental cell line previously used to generate 92 single HLA allele-expressing lines from which we collected mono-allelic MHC I immunopeptidome data. We developed a hierarchical approach to identify translated novel unannotated open reading frames that leverages the large amount of Ribo-seq data we have generated in order to uncover lowly expressed nuORFs, while also preserving tissue specificity. We constructed a database of novel unannotated ORFs (nuORFdb) and used it to analyze mass spectrometry datasets of MHC I-bound peptides, where we detected peptides from 3,555 nuORFs. Additionally, we used nuORFdb to identify cancer-specific nuORFs, as well as nuORFs harboring cancer-specific somatic variants as potential sources of neoantigens in cancer. *This repository contains data from the publicly available cell lines used in this study, including B721.221 cells, A375 cells, HCT116 cells and primary healthy melanocytes (Thermo C0025C). The data pertaining to primary patient samples that are part of this study is deposited in dbGaP.
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2021-11-12 | E-MTAB-10805 | biostudies-arrayexpress
Project description:Mass spectrometry peptidomics coupled with RNA deep sequencing has revealed a large number of translated eukaryotic short open reading frames that produce genomically encoded peptides. The human LOC550643 gene, previously annotated as non-coding, encodes such a peptide (referred to as NoBody). NoBody peptide binds to EDC4 (enhancer of decapping 4) site-specifically, enriches complexes of proteins involved in 5'-3' mRNA degradation, and localizes to P-bodies, which are cellular RNA-protein granules associated with RNA degradation. Furthermore, modulating NoBody expression levels inversely regulates P-body numbers. This peptide has the potential to reveal new insights into the P-body structure-function relationship and cellular regulation of mRNA degradation. We used siRNA-mediated gene silencing coupled with biological pathway analysis to determine the cellular functions of NoBody (LOC550643), and whether it has similar cellular functions to related (Dcp2) and unrelated (DcpS) mRNA degradation proteins. HEK293T cells were silenced for NoBody/LOC550643 expression by transfection with a pool of 4 commercially designed siRNAs (Qiagen). The negative control/reference sample consisted of HEK293T cells transfected with a non-targeting siRNA. Comparison samples were silenced for Dcp2 expression (expected to be functionally related in mRNA decapping and degradation, positive control), DcpS (another mRNA degradation protein from a different pathway, expected to be unrelated, negative control) or GAPDH (unrelated protein, negative control), so that the specificity of cellular responses to NoBody/LOC550643 silencing could be assessed. 48 hours after transfection with siRNA, cells were treated with actinomycin D for 4 hours (expected to blunt transcriptional responses to assay mRNA degradation phenotypes), then harvested and total RNA was extracted and subjected to Affymetrix GeneChip analysis. We note that the mRNA degradation phenotype was apparently overwhelmed by transcriptional responses despite the actinomycin D treatment, so the most meaningful analyses of the data involved gene ontology/biological pathway analysis.
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