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G3 (Bethesda, Md.) Mar 2022Understanding hibernation in brown bears (Ursus arctos) can provide insight into some human diseases. During hibernation, brown bears experience periods of insulin...
Understanding hibernation in brown bears (Ursus arctos) can provide insight into some human diseases. During hibernation, brown bears experience periods of insulin resistance, physical inactivity, extreme bradycardia, obesity, and the absence of urine production. These states closely mimic aspects of human diseases such as type 2 diabetes, muscle atrophy, as well as renal and heart failure. The reversibility of these states from hibernation to active season enables the identification of mediators with possible therapeutic value for humans. Recent studies have identified genes and pathways that are differentially expressed between active and hibernation seasons in bears. However, little is known about the role of differential expression of gene isoforms on hibernation physiology. To identify both distinct and novel mRNA isoforms, full-length RNA-sequencing (Iso-Seq) was performed on adipose, skeletal muscle, and liver from three individual bears sampled during both active and hibernation seasons. The existing reference genome annotation was improved by combining it with the Iso-Seq data. Short-read RNA-sequencing data from six individuals were mapped to the new reference annotation to quantify differential isoform usage (DIU) between tissues and seasons. We identified differentially expressed isoforms in all three tissues, to varying degrees. Adipose had a high level of DIU with isoform switching, regardless of whether the genes were differentially expressed. Our analyses revealed that DIU, even in the absence of differential gene expression, is an important mechanism for modulating genes during hibernation. These findings demonstrate the value of isoform expression studies and will serve as the basis for deeper exploration into hibernation biology.
Topics: Adipose Tissue; Animals; Diabetes Mellitus, Type 2; Gene Expression Regulation; Hibernation; Humans; Protein Isoforms; Ursidae
PubMed: 35100340
DOI: 10.1093/g3journal/jkab422 -
The Journal of Biological Chemistry Jan 2023TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon...
TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.
Topics: Alternative Splicing; Cations; Cell Membrane; Endoplasmic Reticulum; Protein Isoforms; Membrane Proteins; Gene Expression Regulation
PubMed: 36496074
DOI: 10.1016/j.jbc.2022.102781 -
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 -
American Journal of Physiology. Heart... Oct 2022Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα...
Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes. PKCα is an important signaling molecule extensively studied in many cellular processes. However, no isoforms have been reported for PKCα except one prototypic isoform. Alternative mRNA splicing of gene was detected for the first time in rodent and human cardiac tissue, which can produce a previously unknown PKCα-novel exon (NE) isoform. The biochemistry and molecular effects of PKCα-NE are markedly different from PKCα wild type, suggesting potential functional diversity of PKCα signaling in muscle.
Topics: Adult; Alternative Splicing; Amino Acids; Animals; Humans; Lipids; Mice; Muscle, Skeletal; Myocytes, Cardiac; Protein Isoforms; Protein Kinase C-alpha; Proteomics; RNA, Messenger
PubMed: 36112502
DOI: 10.1152/ajpheart.00304.2022 -
Molecules (Basel, Switzerland) Dec 2021After being rather neglected as a research field in the past, carbonic anhydrase activators (CAAs) were undoubtedly demonstrated to be useful in diverse pharmaceutical... (Review)
Review
After being rather neglected as a research field in the past, carbonic anhydrase activators (CAAs) were undoubtedly demonstrated to be useful in diverse pharmaceutical and industrial applications. They also improved the knowledge of the requirements to selectively interact with a CA isoform over the others and confirmed the catalytic mechanism of this class of compounds. Amino acid and amine derivatives were the most explored in in vitro, in vivo and crystallographic studies as CAAs. Most of them were able to activate human or non-human CA isoforms in the nanomolar range, being proposed as therapeutic and industrial tools. Some isoforms are better activated by amino acids than amines derivatives and the stereochemistry may exert a role. Finally, non-human CAs have been very recently tested for activation studies, paving the way to innovative industrial and environmental applications.
Topics: Amines; Amino Acids; Animals; Carbonic Anhydrases; Enzyme Activation; Enzyme Activators; Humans; Models, Molecular; Protein Isoforms
PubMed: 34885917
DOI: 10.3390/molecules26237331 -
Clinical & Experimental Metastasis Jun 2022Cellular plasticity lies at the core of cancer progression, metastasis, and resistance to treatment. Stemness and epithelial-mesenchymal plasticity in cancer are... (Review)
Review
Cellular plasticity lies at the core of cancer progression, metastasis, and resistance to treatment. Stemness and epithelial-mesenchymal plasticity in cancer are concepts that represent a cancer cell's ability to coopt and adapt normal developmental programs to promote survival and expansion. The cancer stem cell model states that a small subset of cancer cells with stem cell-like properties are responsible for driving tumorigenesis and metastasis while remaining especially resistant to common chemotherapeutic drugs. Epithelial-mesenchymal plasticity describes a cancer cell's ability to transition between epithelial and mesenchymal phenotypes which drives invasion and metastasis. Recent research supports the existence of stable epithelial/mesenchymal hybrid phenotypes which represent highly plastic states with cancer stem cell characteristics. The cell adhesion molecule CD44 is a widely accepted marker for cancer stem cells, and it lies at a functional intersection between signaling networks regulating both stemness and epithelial-mesenchymal plasticity. CD44 expression is complex, with alternative splicing producing many isoforms. Interestingly, not only does the pattern of isoform expression change during transitions between epithelial and mesenchymal phenotypes in cancer, but these isoforms have distinct effects on cell behavior including the promotion of metastasis and stemness. The role of CD44 both downstream and upstream of signaling pathways regulating epithelial-mesenchymal plasticity and stemness make this protein a valuable target for further research and therapeutic intervention.
Topics: Alternative Splicing; Epithelial-Mesenchymal Transition; Humans; Hyaluronan Receptors; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Protein Isoforms
PubMed: 35023031
DOI: 10.1007/s10585-022-10146-x -
BMC Biology Oct 2023Through alternative splicing, most human genes produce multiple isoforms in a cell-, tissue-, and disease-specific manner. Numerous studies show that alternative...
BACKGROUND
Through alternative splicing, most human genes produce multiple isoforms in a cell-, tissue-, and disease-specific manner. Numerous studies show that alternative splicing is essential for development, diseases, and their treatments. Despite these important examples, the extent and biological relevance of splicing are currently unknown.
RESULTS
To solve this problem, we developed pairedGSEA and used it to profile transcriptional changes in 100 representative RNA-seq datasets. Our systematic analysis demonstrates that changes in splicing, on average, contribute to 48.1% of the biological signal in expression analyses. Gene-set enrichment analysis furthermore indicates that expression and splicing both convey shared and distinct biological signals.
CONCLUSIONS
These findings establish alternative splicing as a major regulator of the human condition and suggest that most contemporary RNA-seq studies likely miss out on critical biological insights. We anticipate our results will contribute to the transition from a gene-centric to an isoform-centric research paradigm.
Topics: Humans; Gene Expression Profiling; RNA Splicing; Alternative Splicing; RNA-Seq; Sequence Analysis, RNA; Protein Isoforms
PubMed: 37858135
DOI: 10.1186/s12915-023-01724-w -
Experimental Biology and Medicine... Nov 2019We provide a review of Spectrin isoform function in the cytoplasm, the nucleus, the cell surface, and in intracellular signaling. We then discuss the importance of... (Review)
Review
UNLABELLED
We provide a review of Spectrin isoform function in the cytoplasm, the nucleus, the cell surface, and in intracellular signaling. We then discuss the importance of Spectrin’s E2/E3 chimeric ubiquitin conjugating and ligating activity in maintaining cellular homeostasis. Finally we present spectrin isoform subunit specific human diseases. We have created the Spectrinome, from the Human Proteome, Human Reactome and Human Atlas data and demonstrated how it can be a useful tool in visualizing and understanding spectrins myriad of cellular functions.
IMPACT STATEMENT
Spectrin was for the first 12 years after its discovery thought to be found only in erythrocytes. In 1981, Goodman and colleagues found that spectrin-like molecules were ubiquitously found in non-erythroid cells leading to a great multitude of publications over the next thirty eight years. The discovery of multiple spectrin isoforms found associated with every cellular compartment, and representing 2-3% of cellular protein, has brought us to today’s understanding that spectrin is a scaffolding protein, with its own E2/E3 chimeric ubiquitin conjugating ligating activity that is involved in virtually every cellular function. We cover the history, localized functions of spectrin isoforms, human diseases caused by mutations, and provide the spectrinome: a useful tool for understanding the myriad of functions for one of the most important proteins in all eukaryotic cells.
Topics: Animals; Cytoplasm; Humans; Nuclear Matrix-Associated Proteins; Protein Isoforms; Proteomics; Spectrin
PubMed: 31483159
DOI: 10.1177/1535370219867269 -
International Journal of Molecular... Jun 2022Thyroid hormones (THs) control a wide range of physiological functions essential for metabolism, growth, and differentiation. On a molecular level, TH action is exerted... (Review)
Review
Thyroid hormones (THs) control a wide range of physiological functions essential for metabolism, growth, and differentiation. On a molecular level, TH action is exerted by nuclear receptors (TRs), which function as ligand-dependent transcription factors. Among several TR isoforms, the function of TRα2 remains poorly understood as it is a splice variant of TRα with an altered C-terminus that is unable to bind T3. This review highlights the molecular characteristics of TRα2, proposed mechanisms that regulate alternative splicing and indications pointing towards an antagonistic function of this TR isoform in vitro and in vivo. Moreover, remaining knowledge gaps and major challenges that complicate TRα2 characterization, as well as future strategies to fully uncover its physiological relevance, are discussed.
Topics: Alternative Splicing; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Receptors, Thyroid Hormone; Thyroid Hormones
PubMed: 35806002
DOI: 10.3390/ijms23136998 -
Clinical Immunology (Orlando, Fla.) Apr 2022The transcription factor FOXP3 is essential for CD4FOXP3 regulatory T (Treg) cell development and function. Human FOXP3 exists in distinct isoforms and alterations in...
The transcription factor FOXP3 is essential for CD4FOXP3 regulatory T (Treg) cell development and function. Human FOXP3 exists in distinct isoforms and alterations in isoform expression is associated with inflammatory disease progression, however, the exact functions of FOXP3 isoforms remain poorly understood. Herein we used flow cytometry and RNA-sequencing to analyze subsets of Treg cells from two IPEX patients, and a healthy carrier, of a recently described FOXP3 mutation (c.305delT). This mutation is located in exon 2 and results in the loss of the full-length FOXP3 isoform. Treg cells lacking full-length FOXP3 are found at lower-than-expected frequencies. This loss cannot be explained solely by altered thymic output, changes in proliferation, peripheral induction of Treg cells, or apoptosis. Instead, fulllength FOXP3 control a distinct genetic program, involving the previously identified FOXP3 regulators ID3, BCL6 and eIF4E, that upholds Treg cell lineage stability, while it appears nonessential for Treg cell activation.
Topics: Exons; Forkhead Transcription Factors; Gene Expression Regulation; Humans; Protein Isoforms; T-Lymphocytes, Regulatory
PubMed: 35247545
DOI: 10.1016/j.clim.2022.108957