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Lab on a Chip Jun 2021Protein isoforms play a key role in disease progression and arise from mechanisms involving multiple molecular subtypes, including DNA, mRNA and protein. Recently...
Protein isoforms play a key role in disease progression and arise from mechanisms involving multiple molecular subtypes, including DNA, mRNA and protein. Recently introduced multimodal assays successfully link genomes and transcriptomes to protein expression landscapes. However, the specificity of the protein measurement relies on antibodies alone, leading to major challenges when measuring different isoforms of the same protein. Here we utilize microfluidic design to perform same-cell profiling of DNA, mRNA and protein isoforms (triBlot) on low starting cell numbers (1-100 s of cells). After fractionation lysis, cytoplasmic proteins are resolved by molecular mass during polyacrylamide gel electrophoresis (PAGE), adding a degree of specificity to the protein measurement, while nuclei are excised from the device in sections termed "gel pallets" for subsequent off-chip nucleic acid analysis. By assaying TurboGFP-transduced glioblastoma cells, we observe a strong correlation between protein expression prior to lysis and immunoprobed protein. We measure both mRNA and DNA from retrieved nuclei, and find that mRNA levels correlate with protein abundance in TurboGFP-expressing cells. Furthermore, we detect the presence of TurboGFP isoforms differing by an estimated <1 kDa in molecular mass, demonstrating the ability to discern different proteoforms with the same antibody probe. By directly relating nucleic acid modifications to protein isoform expression in 1-100 s of cells, the triBlot assay holds potential as a screening tool for novel biomarkers in diseases driven by protein isoform expression.
Topics: Cell Count; DNA; Electrophoresis, Polyacrylamide Gel; Protein Isoforms; Proteomics
PubMed: 33978041
DOI: 10.1039/d1lc00073j -
Nucleic Acids Research Dec 2023Cell autonomous responses to intracellular bacteria largely depend on reorganization of gene expression. To gain isoform-level resolution of these modes of regulation,...
Cell autonomous responses to intracellular bacteria largely depend on reorganization of gene expression. To gain isoform-level resolution of these modes of regulation, we combined long- and short-read transcriptomic analyses of the response of intestinal epithelial cells to infection by the foodborne pathogen Listeria monocytogenes. Among the most striking isoform-based types of regulation, expression of the cellular stress response regulator CIRBP (cold-inducible RNA-binding protein) and of several SRSFs (serine/arginine-rich splicing factors) switched from canonical transcripts to nonsense-mediated decay-sensitive isoforms by inclusion of 'poison exons'. We showed that damage to host cell membranes caused by bacterial pore-forming toxins (listeriolysin O, perfringolysin, streptolysin or aerolysin) led to the dephosphorylation of SRSFs via the inhibition of the kinase activity of CLK1, thereby driving CIRBP alternative splicing. CIRBP isoform usage was found to have consequences on infection, since selective repression of canonical CIRBP reduced intracellular bacterial load while that of the poison exon-containing isoform exacerbated it. Consistently, CIRBP-bound mRNAs were shifted towards stress-relevant transcripts in infected cells, with increased mRNA levels or reduced translation efficiency for some targets. Our results thus generalize the alternative splicing of CIRBP and SRSFs as a common response to biotic or abiotic stresses by extending its relevance to the context of bacterial infection.
Topics: Humans; Alternative Splicing; Listeriosis; Protein Isoforms; RNA-Binding Proteins; Listeria monocytogenes
PubMed: 37941135
DOI: 10.1093/nar/gkad1033 -
Human Genetics Sep 2017Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the... (Review)
Review
Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as "noise". Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels.
Topics: Animals; Gene Expression Regulation; Humans; Introns; Models, Genetic; Protein Biosynthesis; Protein Isoforms; RNA Stability
PubMed: 28391524
DOI: 10.1007/s00439-017-1791-x -
Protein & Cell May 2011Thyroid hormone plays pivotal roles in growth, differentiation, development and metabolic homeostasis via thyroid hormone receptors (TRs) by controlling the expression... (Review)
Review
Thyroid hormone plays pivotal roles in growth, differentiation, development and metabolic homeostasis via thyroid hormone receptors (TRs) by controlling the expression of TR target genes. The transcriptional activity of TRs is modulated by multiple factors including various TR isoforms, diverse thyroid hormone response elements, different heterodimeric partners, coregulators, and the cellular location of TRs. In the present review, we summarize recent advance in understanding the molecular mechanisms of thyroid hormone action obtained from human subject research, thyroid hormone mimetics application, TR isoform-specific knock-in mouse models, and mitochondrion study with highlights in metabolic regulations. Finally, as future perspectives, we share our thoughts about current challenges and possible approaches to promote our knowledge of thyroid hormone action in metabolism.
Topics: Animals; Gene Knockout Techniques; Humans; Mice; Mitochondria; Protein Isoforms; Receptors, Thyroid Hormone; Thyroid Diseases; Thyroid Hormones
PubMed: 21614672
DOI: 10.1007/s13238-011-1046-x -
Trends in Neurosciences Dec 2009Enhanced acute tolerance predicts alcohol abuse. We describe work on the role of the calcium- and voltage-gated BK channel in alcohol tolerance, highlighting the lipid... (Review)
Review
Enhanced acute tolerance predicts alcohol abuse. We describe work on the role of the calcium- and voltage-gated BK channel in alcohol tolerance, highlighting the lipid environment, BK protein isoform selection and auxiliary BK channel proteins. We show how ethanol, which had the reputation of a nonspecific membrane perturbant, is now being examined at realistic concentrations with cutting-edge techniques, providing novel molecular targets for therapeutic approaches to alcoholism. Addictive disorders impact our emotional, physical and financial status, and burden our healthcare system. Although alcohol is the focus of this review, it is highly probable, given the common neural and biochemical pathways used by drugs of abuse, that the findings described here will also apply to other drugs.
Topics: Alcohol Drinking; Animals; Drug Tolerance; Epigenesis, Genetic; Ethanol; Humans; Large-Conductance Calcium-Activated Potassium Channels; Membrane Lipids; Mice; Mice, Knockout; MicroRNAs; Models, Molecular; Models, Neurological; Neurons; Phosphorylation; Protein Isoforms; Substance-Related Disorders
PubMed: 19781792
DOI: 10.1016/j.tins.2009.08.001 -
Bioinformatics (Oxford, England) Apr 2023Advances in RNA sequencing technologies have achieved an unprecedented accuracy in the quantification of mRNA isoforms, but our knowledge of isoform-specific functions...
MOTIVATION
Advances in RNA sequencing technologies have achieved an unprecedented accuracy in the quantification of mRNA isoforms, but our knowledge of isoform-specific functions has lagged behind. There is a need to understand the functional consequences of differential splicing, which could be supported by the generation of accurate and comprehensive isoform-specific gene ontology annotations.
RESULTS
We present isoform interpretation, a method that uses expectation-maximization to infer isoform-specific functions based on the relationship between sequence and functional isoform similarity. We predicted isoform-specific functional annotations for 85 617 isoforms of 17 900 protein-coding human genes spanning a range of 17 430 distinct gene ontology terms. Comparison with a gold-standard corpus of manually annotated human isoform functions showed that isoform interpretation significantly outperforms state-of-the-art competing methods. We provide experimental evidence that functionally related isoforms predicted by isoform interpretation show a higher degree of domain sharing and expression correlation than functionally related genes. We also show that isoform sequence similarity correlates better with inferred isoform function than with gene-level function.
AVAILABILITY AND IMPLEMENTATION
Source code, documentation, and resource files are freely available under a GNU3 license at https://github.com/TheJacksonLaboratory/isopretEM and https://zenodo.org/record/7594321.
Topics: Humans; Motivation; Protein Isoforms; Software; Alternative Splicing; Sequence Analysis, RNA
PubMed: 36929917
DOI: 10.1093/bioinformatics/btad132 -
Genes & Development Mar 2022Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that is a vital regulator of adipogenesis, insulin sensitivity, and lipid metabolism....
Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that is a vital regulator of adipogenesis, insulin sensitivity, and lipid metabolism. Activation of PPARγ by antidiabetic thiazolidinediones (TZD) reverses insulin resistance but also leads to weight gain that limits the use of these drugs. There are two main PPARγ isoforms, but the specific functions of each are not established. Here we generated mouse lines in which endogenous PPARγ1 and PPARγ2 were epitope-tagged to interrogate isoform-specific genomic binding, and mice deficient in either PPARγ1 or PPARγ2 to assess isoform-specific gene regulation. Strikingly, although PPARγ1 and PPARγ2 contain identical DNA binding domains, we uncovered isoform-specific genomic binding sites in addition to shared sites. Moreover, PPARγ1 and PPARγ2 regulated a different set of genes in adipose tissue depots, suggesting distinct roles in adipocyte biology. Indeed, mice with selective deficiency of PPARγ1 maintained body temperature better than wild-type or PPARγ2-deficient mice. Most remarkably, although TZD treatment improved glucose tolerance in mice lacking either PPARγ1 or PPARγ2, the PPARγ1-deficient mice were protected from TZD-induced body weight gain compared with PPARγ2-deficient mice. Thus, PPARγ isoforms have specific and separable metabolic functions that may be targeted to improve therapy for insulin resistance and diabetes.
Topics: Adipocytes; Animals; Gene Expression Regulation; Insulin Resistance; Mice; PPAR gamma; Protein Isoforms; Thiazolidinediones
PubMed: 35273075
DOI: 10.1101/gad.349232.121 -
Nucleic Acids Research Jan 2018The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms...
The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms in protein-coding genes. APPRIS selects a single protein isoform, the 'principal' isoform, as the reference for each gene based on these annotations. A single main splice isoform reflects the biological reality for most protein coding genes and APPRIS principal isoforms are the best predictors of these main proteins isoforms. Here, we present the updates to the database, new developments that include the addition of three new species (chimpanzee, Drosophila melangaster and Caenorhabditis elegans), the expansion of APPRIS to cover the RefSeq gene set and the UniProtKB proteome for six species and refinements in the core methods that make up the annotation pipeline. In addition APPRIS now provides a measure of reliability for individual principal isoforms and updates with each release of the GENCODE/Ensembl and RefSeq reference sets. The individual GENCODE/Ensembl, RefSeq and UniProtKB reference gene sets for six organisms have been merged to produce common sets of splice variants.
Topics: Alternative Splicing; Amino Acid Sequence; Animals; Databases, Genetic; Humans; Models, Molecular; Molecular Sequence Annotation; Protein Conformation; Protein Isoforms; Proteome; Reproducibility of Results; Sequence Alignment
PubMed: 29069475
DOI: 10.1093/nar/gkx997 -
Cell Reports Mar 2024The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two,...
The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two, WDR33v2 (V2) and WDR33v3 (V3), have not been studied. Here, we report that V2 and V3 are generated by alternative polyadenylation, and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly, V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING), the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-β induction by preventing STING disulfide oligomerization but promotes autophagy, likely by recruiting WIPI2 isoforms. V3, on the other hand, functions to increase STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated, indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated.
Topics: Humans; mRNA Cleavage and Polyadenylation Factors; RNA, Messenger; Polyadenylation; Membrane Proteins; Protein Isoforms; Immunity, Innate
PubMed: 38430516
DOI: 10.1016/j.celrep.2024.113886 -
Proteomics Oct 2012Protein isoforms/splice variants can play important roles in various biological processes and can potentially be used as biomarkers or therapeutic targets/mediators.... (Review)
Review
Protein isoforms/splice variants can play important roles in various biological processes and can potentially be used as biomarkers or therapeutic targets/mediators. Thus, there is a need for efficient and, importantly, accurate methods to distinguish and quantify specific protein isoforms. Since protein isoforms can share a high percentage of amino acid sequence homology and dramatically differ in their cellular concentration, the task for accuracy and efficiency in methodology and instrumentation is challenging. The analysis of intact proteins has been perceived to provide a more accurate and complete result for isoform identification/quantification in comparison to analysis of the corresponding peptides that arise from protein enzymatic digestion. Recently, novel approaches have been explored and developed that can possess the accuracy and reliability important for protein isoform differentiation and isoform-specific peptide targeting. In this review, we discuss the recent development in methodology and instrumentation for enhanced detection of protein isoforms as well as the examples of their biological importance.
Topics: Amino Acid Sequence; Animals; Chromatography, Liquid; Humans; Mass Spectrometry; Molecular Sequence Data; Protein Isoforms; Proteomics; Sequence Alignment
PubMed: 22888084
DOI: 10.1002/pmic.201200161