Dataset Information

Identification of Alternative Splicing in Proteomes of Human Melanoma Cell Lines without RNA Sequencing Data.

ABSTRACT: Alternative splicing is one of the main regulation pathways in living cells beyond simple changes in the level of protein expression. Most of the approaches proposed in proteomics for the identification of specific splicing isoforms require a preliminary deep transcriptomic analysis of the sample under study, which is not always available, especially in the case of the re-analysis of previously acquired data. Herein, we developed new algorithms for the identification and validation of protein splice isoforms in proteomic data in the absence of RNA sequencing of the samples under study. The bioinformatic approaches were tested on the results of proteome analysis of human melanoma cell lines, obtained earlier by high-resolution liquid chromatography and mass spectrometry (LC-MS). A search for alternative splicing events for each of the cell lines studied was performed against the database generated from all known transcripts (RefSeq) and the one composed of peptide sequences, which included all biologically possible combinations of exons. The identifications were filtered using the prediction of both retention times and relative intensities of fragment ions in the corresponding mass spectra. The fragmentation mass spectra corresponding to the discovered alternative splicing events were additionally examined for artifacts. Selected splicing events were further validated at the mRNA level by quantitative PCR.

SUBMITTER: Lobas AA

PROVIDER: S-EPMC9916885 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Identification of Alternative Splicing in Proteomes of Human Melanoma Cell Lines without RNA Sequencing Data.

Lobas Anna A AA Solovyeva Elizaveta M EM Levitsky Lev I LI Goncharov Anton O AO Lyssuk Elena Y EY Larin Sergey S SS Moshkovskii Sergei A SA Gorshkov Mikhail V MV

International journal of molecular sciences 20230127 3

Alternative splicing is one of the main regulation pathways in living cells beyond simple changes in the level of protein expression. Most of the approaches proposed in proteomics for the identification of specific splicing isoforms require a preliminary deep transcriptomic analysis of the sample under study, which is not always available, especially in the case of the re-analysis of previously acquired data. Herein, we developed new algorithms for the identification and validation of protein sp ...[more]

PMID: 36768787

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Project description:Paratuberculosis is a major endemic disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and leads to huge economic loss in the dairy sector worldwide. Alternative splicing (AS) events, playing indispensable regulatory roles in many protein functions and biological pathways, are shown to be associated with complex traits and diseases. In this study, by integrating the RNA sequencing (RNA-seq) data of 24 samples from three tissues (peripheral blood, jejunum and salivary gland) of Holstein cows, we obtained 2,706,541,696 uniquely mapped reads in total that represented 12,870 expressed genes, and detected 4285 differentially expressed genes (DEGs) between MAP-infected and healthy cows (p < 0.05). Of them, 92 differentially expressed splicing factors (DESFs) were included. Further, 119, 150 and 68 differential alternative splicing (DAS) events between MAP-infected and healthy cows were identified in peripheral blood, jejunum and salivary glands, respectively. Of note, six DAS events were highly and significantly correlated with the DESFs (R2 > 0.9; p < 0.01), and their corresponding genes (COPI coat complex subunit gamma 2gene (COPG2), kinesin family member 2C gene (KIF2C), exocyst complex component 7 (EXOC7), Rab9 effector protein with kelch motifs gene (RABEPK), deoxyribonuclease 1 gene (DNASE1) and early endosome antigen 1gene (EEA1)) were significantly enriched in immune response such as vesicle-mediated transport, regulation of acute inflammatory response and tuberculosis through gene ontology (GO) and KEGG analysis. KS test showed that the DAS events in the EXOC7 and KIF2C genes indeed displayed differences between MAP-infected cows and healthy cows. The DAS in EXOC7 might produce a new protein sequence with lack of 23 amino acids, and the DAS in KIF2C induced a stop codon of premature occurrence and resulted in a lack of functional domain. In summary, this study identified the DAS events and corresponding genes related to MAP-infection base on the RNA-seq data from multiple tissues of Holstein cows, providing novel insights into the regulatory mechanisms underpinning paratuberculosis in dairy cattle.

Project description:Alternative splicing (AS) regulates multiple biological processes including flowering, circadian and stress response in plant. Although accumulating evidences indicate that AS is developmentally regulated, how AS responds to developmental cues is not well understood. Early fruit growth mainly characterized by active cell division and cell expansion contributes to the formation of fruit morphology and quality traits. Transcriptome profiling has revealed the coordinated complex regulation of gene expression in the process. High throughput RNA sequencing (RNA-seq) technology is advancing the genome-wide analysis of AS events in plant species, but the landscape of AS in early growth fruit is still not available for tomato (Solanum lycopersicum), a model plant for fleshy fruit development study.Using RNA-seq, we surveyed the AS patterns in tomato seedlings, flowers and young developing fruits and found that 59.3 % of expressed multi-exon genes underwent AS in these tissues. The predominant type of AS events is intron retention, followed by alternative splice donor and acceptor, whereas exon skipping has the lowest frequency. Although the frequencies of AS events are similar among seedlings, flowers and early growth fruits, the fruits generated more splice variants per gene. Further comparison of gene expression in early growth fruits at 2, 5 and 10 days post anthesis revealed that 5206 multi-exon genes had at least one splice variants differentially expressed during early fruit development, whereas only 1059 out of them showed differential expression at gene level. We also identified 27 multi-exon genes showing differential splicing during early fruit growth. In addition, the study discovered 2507 new transcription regions (NTRs) unlinked to the annotated chromosomal regions, from where 956 putative protein coding transcripts and 1690 putative long non-coding RNAs were identified.Our genome-wide analysis of AS events reveals a distinctive AS pattern in early growth tomato fruits. The landscape of AS obtained in this study will facilitate future investigation on transcriptome complexity and AS regulation during early fruit growth in tomato. The newly found NTRs will also be useful for updating the tomato genome annotation.

Dataset Information

Identification of Alternative Splicing in Proteomes of Human Melanoma Cell Lines without RNA Sequencing Data.

Publications

Identification of Alternative Splicing in Proteomes of Human Melanoma Cell Lines without RNA Sequencing Data.

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