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STAR Protocols Dec 2020Alternative splicing greatly expands the coding capacity of the human genome, but how many alternative transcripts are translated as proteins or carry functional...
Alternative splicing greatly expands the coding capacity of the human genome, but how many alternative transcripts are translated as proteins or carry functional importance remains unknown and awaits experimental verification. Here, we describe a protocol that combines transcriptomics (RNA-seq) and proteomics (mass spectrometry [MS]) analyses to identify alternative isoforms in proteomes. This workflow is applicable to custom-generated RNA-seq and MS data from matching samples, as well as the reanalysis of existing transcriptomics and proteomics datasets in public repositories. For complete details on the use and execution of this protocol, please refer to Lau et al. (2019).
Topics: Alternative Splicing; Base Sequence; Gene Expression; Gene Expression Profiling; Humans; Mass Spectrometry; Protein Isoforms; Proteome; Proteomics; RNA-Seq; Sequence Analysis, RNA; Tandem Mass Spectrometry; Transcriptome
PubMed: 33377032
DOI: 10.1016/j.xpro.2020.100138 -
BMC Genomics 2015Protein-protein interactions (PPIs) are key to understanding diverse cellular processes and disease mechanisms. However, current PPI databases only provide...
BACKGROUND
Protein-protein interactions (PPIs) are key to understanding diverse cellular processes and disease mechanisms. However, current PPI databases only provide low-resolution knowledge of PPIs, in the sense that "proteins" of currently known PPIs generally refer to "genes." It is known that alternative splicing often impacts PPI by either directly affecting protein interacting domains, or by indirectly impacting other domains, which, in turn, impacts the PPI binding. Thus, proteins translated from different isoforms of the same gene can have different interaction partners.
RESULTS
Due to the limitations of current experimental capacities, little data is available for PPIs at the resolution of isoforms, although such high-resolution data is crucial to map pathways and to understand protein functions. In fact, alternative splicing can often change the internal structure of a pathway by rearranging specific PPIs. To fill the gap, we systematically predicted genome-wide isoform-isoform interactions (IIIs) using RNA-seq datasets, domain-domain interaction and PPIs. Furthermore, we constructed an III database (IIIDB) that is a resource for studying PPIs at isoform resolution. To discover functional modules in the III network, we performed III network clustering, and then obtained 1025 isoform modules. To evaluate the module functionality, we performed the GO/pathway enrichment analysis for each isoform module.
CONCLUSIONS
The IIIDB provides predictions of human protein-protein interactions at the high resolution of transcript isoforms that can facilitate detailed understanding of protein functions and biological pathways. The web interface allows users to search for IIIs or III network modules. The IIIDB is freely available at http://syslab.nchu.edu.tw/IIIDB.
Topics: Algorithms; Alternative Splicing; Cluster Analysis; Computational Biology; Databases, Protein; Genome, Human; Humans; Internet; Protein Interaction Mapping; Protein Interaction Maps; Protein Isoforms; Proteins; Reproducibility of Results; Software
PubMed: 25707505
DOI: 10.1186/1471-2164-16-S2-S10 -
Cell Death & Disease Mar 2024The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote... (Review)
Review
The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote tumorigenesis. An active area in cancer research is uncovering the roles of ubiquitination on spliceosome assembly contributing to transcript diversity and expression of alternative isoforms. However, the effects of isoform changes on functionality of ubiquitination machineries (E1, E2, E3, E4, and deubiquitinating (DUB) enzymes) influencing onco- and tumor suppressor protein stabilities is currently understudied. Characterizing these changes could be instrumental in improving cancer outcomes via the identification of novel biomarkers and targetable signaling pathways. In this review, we focus on highlighting reported examples of direct, protein-coded isoform variation of ubiquitination enzymes influencing cancer development and progression in gastrointestinal (GI) malignancies. We have used a semi-automated system for identifying relevant literature and applied established systems for isoform categorization and functional classification to help structure literature findings. The results are a comprehensive snapshot of known isoform changes that are significant to GI cancers, and a framework for readers to use to address isoform variation in their own research. One of the key findings is the potential influence that isoforms of the ubiquitination machinery have on oncoprotein stability.
Topics: Humans; Ubiquitination; Protein Isoforms; Gastrointestinal Neoplasms; Carcinogenesis; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases
PubMed: 38453895
DOI: 10.1038/s41419-024-06575-z -
BMC Psychiatry Jul 2012The orbitofrontal cortex (OFC) may play a role in the pathogenesis of psychiatric illnesses such as bipolar disorder and schizophrenia, in which...
BACKGROUND
The orbitofrontal cortex (OFC) may play a role in the pathogenesis of psychiatric illnesses such as bipolar disorder and schizophrenia, in which hypothalamic-pituitary-adrenal (HPA) axis abnormalities are observed and stress has been implicated. A critical component of the HPA axis which mediates cellular stress responses in the OFC, and has been implicated in psychiatric illness, is the glucocorticoid receptor (GR).
METHODS
In the lateral OFC, we employed quantitative real-time PCR and western blotting to investigate GR mRNA and protein expression in 34 bipolar disorder cases, 35 schizophrenia cases and 35 controls. Genotype data for eleven GR gene (NR3C1) polymorphisms was also used to explore possible effects of NR3C1 sequence variation on GR mRNA and protein expression in the lateral OFC.
RESULTS
We found no diagnostic differences in pan GR, GR-1C or GR-1F mRNA expression. However, the GR-1B mRNA transcript variant was decreased (14.3%) in bipolar disorder cases relative to controls (p < 0.05), while GR-1H mRNA was decreased (22.0%) in schizophrenia cases relative to controls (p < 0.005). By western blotting, there were significant increases in abundance of a truncated GRα isoform, putative GRα-D1, in bipolar disorder (56.1%, p < 0.005) and schizophrenia (31.5% p < 0.05). Using genotype data for eleven NR3C1 polymorphisms, we found no evidence of effects of NR3C1 genotype on GR mRNA or GRα protein expression in the OFC.
CONCLUSIONS
These findings reveal selective abnormalities of GR mRNA expression in the lateral OFC in psychiatric illness, which are more specific and may be less influenced by NR3C1 genotype than those of the dorsolateral prefrontal cortex reported previously. Our results suggest that the GRα-D1 protein isoform may be up-regulated widely across the frontal cortex in psychiatric illness.
Topics: Adult; Bipolar Disorder; Female; Frontal Lobe; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Protein Isoforms; RNA, Messenger; Receptors, Glucocorticoid; Schizophrenia; Up-Regulation
PubMed: 22812453
DOI: 10.1186/1471-244X-12-84 -
Nature Communications Sep 2023Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the...
Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to maximize isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive isoform-level characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.
Topics: Humans; Proteomics; Protein Isoforms; Biomedical Research; Carcinoma, Hepatocellular; Liver Neoplasms
PubMed: 37726316
DOI: 10.1038/s41467-023-41558-2 -
Nature Protocols Feb 2021Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not...
Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, 'gel pallets' containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2-3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos.
Topics: Animals; Blastocyst; Blastomeres; DNA; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Embryonic Development; Female; Immunoblotting; Mice; Nucleic Acids; Protein Isoforms; RNA, Messenger
PubMed: 33452502
DOI: 10.1038/s41596-020-00449-2 -
Neuropsychopharmacology : Official... Dec 2011Stress has been implicated in the onset and illness course of schizophrenia and bipolar disorder. The effects of stress in these disorders may be mediated by...
Stress has been implicated in the onset and illness course of schizophrenia and bipolar disorder. The effects of stress in these disorders may be mediated by abnormalities of the hypothalamic-pituitary-adrenal axis, and its corticosteroid receptors. We investigated mRNA expression of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and protein expression of multiple GRα isoforms, in the prefrontal cortex of 37 schizophrenia cases and 37 matched controls. Quantitative real-time PCR, western blotting, and luciferase assays were employed. In multiple regression analysis, schizophrenia diagnosis was a significant predictor of total GR mRNA expression (p<0.05), which was decreased (11.4%) in schizophrenia cases relative to controls. No significant effect of diagnosis on MR mRNA was detected. At the protein level, no significant predictors of total GRα protein or the full-length GRα isoform were identified. However, schizophrenia diagnosis was a strong predictor (p<0.0005) of the abundance of a truncated ≈ 50 kDa GRα protein isoform, putative GRα-D1, which was increased in schizophrenia cases (80.4%) relative to controls. This finding was replicated in a second cohort of 35 schizophrenia cases, 34 bipolar disorder cases, and 35 controls, in which both schizophrenia and bipolar disorder diagnoses were significant predictors of putative GRα-D1 abundance (p<0.05 and p=0.005, respectively). Full-length GRα was increased in bipolar disorder relative to schizophrenia cases. Luciferase assays demonstrated that the GRα-D1 isoform can activate transcription at glucocorticoid response elements. These findings confirm total GR mRNA reductions in schizophrenia and provide the first evidence of GR protein isoform abnormalities in schizophrenia and bipolar disorder.
Topics: Adolescent; Adult; Aged; Bipolar Disorder; Cohort Studies; Down-Regulation; Female; Humans; Male; Middle Aged; Prefrontal Cortex; Protein Isoforms; RNA, Messenger; Receptors, Glucocorticoid; Receptors, Mineralocorticoid; Schizophrenia; Young Adult
PubMed: 21881570
DOI: 10.1038/npp.2011.160 -
Expert Review of Vaccines Apr 2015The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide...
The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy.
Topics: Adjuvants, Immunologic; Biomedical Research; Humans; Interleukin-33; Interleukins; Protein Isoforms; Vaccines
PubMed: 25656504
DOI: 10.1586/14760584.2015.1011135 -
RNA Biology Jan 2022Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be...
Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.[Figure: see text].
Topics: Humans; Proteogenomics; Human Umbilical Vein Endothelial Cells; Protein Isoforms; Alternative Splicing; RNA
PubMed: 36457147
DOI: 10.1080/15476286.2022.2141938 -
British Journal of Cancer Aug 2007p53, p63 and p73 are members of the p53 gene family involved in development, differentiation and response to cellular stress. p53 gene is a transcription factor... (Review)
Review
p53, p63 and p73 are members of the p53 gene family involved in development, differentiation and response to cellular stress. p53 gene is a transcription factor essential for the prevention of cancer formation. The p53 pathway is ubiquitously lost in human cancer either by p53 gene mutation (60% of cancers) or by lost of cell signalling upstream and downstream of p53 in the remaining cancers expressing WTp53 gene. As p53 pathway inactivation is a common denominator to all cancers, the understanding of p53 tumour suppressor activity is likely to bring us closer to cancer therapy. However, despite all the experimental evidences showing the importance of p53 in preventing carcinogenesis, it is difficult in clinical studies to link p53 status to cancer treatment and clinical outcome. The recent discovery that p53 gene encodes for nine different p53 proteins (isoforms) may have a profound impact on our understanding of p53 tumour suppressor activity. Studies in several tumour types have shown that the nine different p53 isoforms are abnormally expressed in tumour tissues compared to normal cells. p53 protein isoforms modulate p53 transcriptional activity and cell fate outcome in response to stress. Regulation of p53 function in normal and tumour tissues in man is likely to be more complex than has been hitherto appreciated. Therefore, the tumour p53 status needs to be determined more accurately by integrating p53 isoform expression, functional p53 mutation analysis and a panel of antibodies specific of p53 and of its target genes.
Topics: Humans; Mutation; Neoplasms; Protein Isoforms; Tumor Suppressor Protein p53
PubMed: 17637683
DOI: 10.1038/sj.bjc.6603886