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Viruses Feb 2023Interferon lambdas (IFNLs) are innate immune cytokines that induce antiviral cellular responses by signaling through a heterodimer composed of IL10RB and the interferon...
Interferon lambdas (IFNLs) are innate immune cytokines that induce antiviral cellular responses by signaling through a heterodimer composed of IL10RB and the interferon lambda receptor 1 (IFNLR1). Multiple transcriptional variants are expressed in vivo and are predicted to encode distinct protein isoforms whose function is not fully established. isoform 1 has the highest relative transcriptional expression and encodes the full-length functional form that supports canonical IFNL signaling. isoforms 2 and 3 have lower relative expression and are predicted to encode signaling-defective proteins. To gain insight into IFNLR1 function and regulation, we explored how altering relative expression of IFNLR1 isoforms influenced the cellular response to IFNLs. To achieve this, we generated and functionally characterized stable HEK293T clones expressing doxycycline-inducible FLAG-tagged IFNLR1 isoforms. Minimal FLAG-IFNLR1 isoform 1 overexpression markedly increased IFNL3-dependent expression of antiviral and pro-inflammatory genes, a phenotype that could not be further augmented by expressing higher levels of FLAG-IFNLR1 isoform 1. Expression of low levels of FLAG-IFNLR1 isoform 2 led to partial induction of antiviral genes, but not pro-inflammatory genes, after IFNL3 treatment, a phenotype that was largely abrogated at higher FLAG-IFNLR1 isoform 2 expression levels. Expression of FLAG-IFNLR1 isoform 3 partially augmented antiviral gene expression after IFNL3 treatment. In addition, FLAG-IFNLR1 isoform 1 significantly reduced cellular sensitivity to the type-I IFN IFNA2 when overexpressed. These results identify a unique influence of canonical and non-canonical IFNLR1 isoforms on mediating the cellular response to interferons and provide insight into possible pathway regulation in vivo.
Topics: Humans; HEK293 Cells; Interferon Lambda; Interferons; Protein Isoforms; Receptors, Interferon; Antiviral Restriction Factors
PubMed: 36992341
DOI: 10.3390/v15030632 -
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 -
Molecular Oncology Jan 2022Breast cancer is the most diagnosed malignancy in women, with over half a million women dying from this disease each year. In our previous studies, ∆40p53, an...
Breast cancer is the most diagnosed malignancy in women, with over half a million women dying from this disease each year. In our previous studies, ∆40p53, an N-terminally truncated p53 isoform, was found to be upregulated in breast cancers, and a high ∆40p53 : p53α ratio was linked with worse disease-free survival. Although p53α inhibits cancer migration and invasion, little is known about the role of ∆40p53 in regulating these metastasis-related processes and its role in contributing to worse prognosis. The aim of this study was to assess the role of ∆40p53 in breast cancer migration and invasion. A relationship between Δ40p53 and gene expression profiles was identified in oestrogen-receptor-positive breast cancer specimens. To further evaluate the role of Δ40p53 in oestrogen-receptor-positive breast cancer, MCF-7 and ZR75-1 cell lines were transduced to knockdown p53α or Δ40p53 and overexpress Δ40p53. Proliferation, migration and invasion were assessed in the transduced sublines, and gene expression was assessed through RNA-sequencing and validated by reverse-transcription quantitative PCR. Knockdown of both p53α and ∆40p53 resulted in increased proliferation, whereas overexpression of ∆40p53 reduced proliferation rates. p53α knockdown was also associated with increased cell mobility. ∆40p53 overexpression reduced both migratory and invasive properties of the transduced cells. Phenotypic findings are supported by gene expression data, including differential expression of LRG1, HYOU1, UBE2QL1, SERPINA5 and PCDH7. Taken together, these results suggest that, at the basal level, ∆40p53 works similarly to p53α in suppressing cellular mobility and proliferation, although the role of Δ40p53 may be cell context-specific.
Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53
PubMed: 34657382
DOI: 10.1002/1878-0261.13118 -
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 -
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 -
Cell Cycle (Georgetown, Tex.) Jul 2020The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections,...
The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections, inflammation and infertility. In addition to this, Trp73 mice display severe alteration in the ciliated epithelial lining and the full-length -terminal isoform TAp73 has been implicated in the control of multiciliogenesis transcriptional program. With our recently generated Trp73 mouse model, we interrogate the physiological role of p73 C-terminal isoforms . Trp73 mice lack exon 13 in Trp73 gene, producing an ectopic switch from the C-terminal isoforms p73α to p73β. Trp73 mice show a pattern of expression of TAp73 comparable to the wild-type littermates, indicating that the to switch does not significantly alter the expression of the gene in this cell type. Moreover, Trp73 do not display any significant alteration in the airway ciliated epithelium, suggesting that in this context p73β can fully substitute the function of the longer isoform p73α. Similarly, Trp73 ciliated epithelium of the brain ependyma also does appear defective. In this district however expression of TAp73 is not detectable, indicating that expression of the gene might be compensated by alternative mechanisms. Overall our work indicates that C-terminus p73 is dispensable for the multiciliogenesis program and suggests a possible tissue-specific effect of p73 alternative splicing.
Topics: Animals; Cell Line; Cilia; Ependyma; Epithelium; Mice; Mitochondria; Organogenesis; Protein Isoforms; Structure-Activity Relationship; Trachea; Tumor Protein p73
PubMed: 32584647
DOI: 10.1080/15384101.2020.1783055 -
The Biochemical Journal Jul 2021Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma...
Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other.
Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calorimetry; Cell Cycle Proteins; Circular Dichroism; DNA-Binding Proteins; Magnetic Resonance Spectroscopy; Peptides; Protein Binding; Protein Isoforms; Protozoan Proteins; Sequence Homology, Amino Acid; Thermodynamics; Toxoplasma; Toxoplasmosis
PubMed: 34114596
DOI: 10.1042/BCJ20210295 -
BMC Nephrology Dec 2023The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft...
The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocin) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocin and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocin and podocin. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocin were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocin, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocin were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.
Topics: Humans; Animals; Mice; Podocytes; Intracellular Signaling Peptides and Proteins; Protein Isoforms; Nephrotic Syndrome; RNA, Messenger
PubMed: 38114895
DOI: 10.1186/s12882-023-03420-x -
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