Mapping start codons of small open reading frames by N-terminomics approach
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Cuihong Wan
PROVIDER: PXD046680 | iProX | Mon Nov 06 00:00:00 GMT 2023
REPOSITORIES: iProX
Similar Datasets
Project description:The C-terminal of proteins regulates their biological functions. Small open reading frame-encoded peptides (SEPs) are a novel class of gene expression products that participate in various biological activities. The C-termini of SEPs have also been found to affect their function, but the polymorphism of SEPs' C-terminal has not yet been systematically elucidated. Here, we utilized C-terminal proteomics technology to systematically analyze the C-termini of SEPs in human tumor cell lines. We combined two SEPs enrichment methods (ACN and heat precipitation) and two C-terminal peptides enrichment methods (MOLEX and CTAILS) to identified the C-terminal peptides of SEPs. Each experiment was performed with triplicate.
2025-06-03 | PXD064597 |
Project description:We identified phosphorylated SEPs in Hep3B cells. ACN precipitation, thermo precipitation and HCl precipitation were used to enrich SEPs. Three materials (nmTiO2, umTiO2, IMAC) were used to enrich phosphorylated peptides.
2024-04-15 | PXD047431 |
Project description:RNA-Seq reads and TopHat (Trapnell et al. Bioinformatics 2009) alignments of K562 cell-line transcriptome. These were used to validate the expression of short peptides idenitified by Mass-Spectrometry in K562 cells. K562 polyA+ RNA (Batch 1) and total RNA (batch 2) was purchased from Ambion. We used oligo (dT)-selected polyA+ RNA to construct libraries for RNA-Seq.We then profiled the transcriptome of polyadenylated mRNA-Seq using Illumina sequencing platforms. We then used the sequenced reads to reconstruct the transcriptome using the Cufflinks de-novo assembler (Trapnell et al. Nat.Bio.Tech. 2010). Recent computational and ribosome profiling analyses suggest that many short open reading frames (sORFs) in eukaryotic genomes are translated. However, evidence that these sORFs produce stable polypeptides is lacking. Here we develop a strategy to discover and validate novel sORF-encoded polypeptides (SEPs) in human cells. In total, we detect 117 SEPs, 114 of which are novel, varying in length from 15 to 149 amino acids. Of these, 10 SEPs (0.5%) are derived from long intergenic non-coding RNAs (lincRNAs). We also observe the presence of polycistronic genes and the widespread use of non-AUG start codons, which is a phenomenon historically thought to be rare in the mammalian genome. Quantitative measurements reveal that SEPs can be found at concentrations between ~10-2000 copies per cell, which is within the range of typical cellular proteins. We confirm the translation of a number of these SEPs through heterologous expression of their encoding cDNAs. We also discover that several SEPs possess properties characteristic of functional proteins. These results demonstrate that human sORFs produce numerous stable polypeptides, revealing that the human proteome is larger and more diverse than previously appreciated.
2012-11-14 | E-GEOD-34740 | biostudies-arrayexpress
Project description:Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
2021-01-11 | GSE164239 | GEO
Project description:Many small open reading frames (smORFs) embedded in lncRNA transcripts have been shown to encode biologically functional polypeptides (smORFs-encoded polypeptides, SEPs) in different organisms. Despite significant advances in genomics, bioinformatics and proteomics that largely enabled the discovery of novel SEPs, their identification across different biological samples is still hampered by their poor predictability, diminutive size and low relative abundance. Here, we take advantage of NONCODE, a repository containing the most complete collection and annotation of lncRNA transcripts from different species, to build a novel database that attempts to maximize a collection of SEPs from human and mouse lncRNA transcripts. In order to further improve SEP discovery, we implemented two effective and complementary polypeptide enrichment strategies, 30 kDa MWCO filter and C8 SPE column. These combined strategies enabled us to discover 357 and 409 SEPs from, respectively, 8 human cell lines, and 3 mouse cell lines and 8 mouse tissues. Importantly, nineteen of the identified SEPs were then verified through in-vitro expression, immunoblotting, parallel reaction monitoring (PRM) and synthetic peptides. Subsequent bioinformatic analysis revealed that some of the physical and chemical properties of these novel SEPs, including amino acid composition and codon usage, are different from those commonly found in canonical proteins. Intriguingly, nearly 65% of the identified SEPs were found to be initiated with non-AUG start codons. Overall, the strategy presented in this study encompasses an efficient workflow that enabled us to identify 766 novel SEPs across multiple cell lines and tissues, which probably represents the largest number of SEPs detected by mass spectrometry reported to date. These novel SEPs might not only provide new clues for the annotation of noncoding elements in the genome but can also serve as a valuable resource for the functional characterization of individual SEPs.
2021-06-15 | PXD019486 | Pride
Project description:RNA co-immunoprecipitations with C-terminal protein A-tagged She proteins. One liter of cells were cultured at 30 degrees in YPAD medium and collected during exponential growth by centrifugation. Cells were washed twice and broken mechanically with glass beads. Extracts were incubated with IgG-agarose beads (Sigma). The beads were washed four times, and She proteins were released from the beads by cleavage with TEV-protease (Invitrogen). RNA was isolated by phenol/chloroform extraction and isopropanol precipitation from TEV eluates, which corresponds to the purified fraction, and from extracts (input). Both RNA samples, input and purified, were reverse transcribed and labeled with the fluorescent dyes Cy3 and Cy5 (Amersham), respectively. The samples were mixed and competitively hybridized to yeast DNA microarrays containing all yeast genes
2005-10-14 | E-SMDB-2092 | biostudies-arrayexpress
Project description:Long non-coding RNAs (lncRNAs) are generally defined as RNA transcripts longer than 200 nucleotides that are not translated into proteins. Recently, many small open reading frames (smORFs) embedded in lncRNA scripts have been verified to be able to encode functional polypeptides (namely lncRNA-SEPs here). Although collaborative analysis by advanced genomics, bioinformatics and proteomics largely drives SEPs discovery, the poor predictability, diminutive size and low abundance still challenge systematic identification of SEPs from different biological samples. Here, we took advantage of the NONCODE database that deposited with the most complete collection and annotation of lncRNA transcripts from different species to build a database that to maximally collect all putative small ORFs from human and mouse lncRNA transcripts. Two effective and complementary polypeptides enrichment strategies (30 kDa MWCO filter and C8 SPE column) were also integrated to further improve the discovery of novel lncRNA-SEPs. These efforts led to the discovery of 362 lncRNA-SEPs from 8 human cell lines and 238 lncRNA-SEPs from 3 mouse cell lines and 8 mouse tissues. 18 out of these lncRNA-SEPs were verified experimentally by multiple technologies including in vitro expression, immunoblotting and parallel reaction monitoring-based mass spectrometry (PRM-MS) in 293T cells. Further bioinformatic analysis reveals that the physical and chemical properties of these novel lncRNA-SEPs, such as amino acid composition and codon usage, are varied from canonical proteins. Intriguingly, nearly 70% of the identified lncRNA-SEPs were found to be initiated with non-AUG start codons. Collectively, the efficient workflows presented in this study enables us identify 600 novel lncRNA-SPEs from multiple cell lines and tissues, which should represent the largest number of MS-detected lncRNA-encoding SEPs ever reported to date. These novel lncRNA-SEPs not only could provide new clues for the annotation of the noncoding elements in the genome, but also could serve as a valuable resource for the functional characterization of individual lncRNA-SEPs.
2023-05-06 | PXD016981 | Pride
Project description:Computational, genomic, and proteomic approaches have been used to discover nonannotated proteincoding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs.
2019-06-26 | MSV000084014 | MassIVE
Project description:RNA co-immunoprecipitations with C-terminal protein A-tagged She proteins. One liter of cells were cultured at 30 degrees in YPAD medium and collected during exponential growth by centrifugation. Cells were washed twice and broken mechanically with glass beads. Extracts were incubated with IgG-agarose beads (Sigma). The beads were washed four times, and She proteins were released from the beads by cleavage with TEV-protease (Invitrogen). RNA was isolated by phenol/chloroform extraction and isopropanol precipitation from TEV eluates, which corresponds to the purified fraction, and from extracts (input). Both RNA samples, input and purified, were reverse transcribed and labeled with the fluorescent dyes Cy3 and Cy5 (Amersham), respectively. The samples were mixed and competitively hybridized to yeast DNA microarrays containing all yeast genes Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-12-21 | GSE3879 | GEO
Project description:Established MOC22 tumors in WT B6 mice were treated with three intratumoral injections of 0.4 ug NHS-rmIL12; control and treated tumors were analyzed for immune correlative changes with single-cell RNA-seq.
2022-04-13 | GSE186636 | GEO