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G3 (Bethesda, Md.) Nov 2022Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read...
Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.
Topics: Humans; Male; Transcriptome; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Protein Isoforms; Sequence Analysis, RNA; Lymphocyte Subsets
PubMed: 36161486
DOI: 10.1093/g3journal/jkac253 -
Acta Pharmacologica Sinica Oct 2015The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However,... (Review)
Review
The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents.
Topics: Animals; Antineoplastic Agents; Humans; Molecular Targeted Therapy; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Isoforms; Protein Kinase Inhibitors; Purines; Quinazolinones
PubMed: 26364801
DOI: 10.1038/aps.2015.71 -
Nucleosides, Nucleotides & Nucleic Acids 2022Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic...
Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of , i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of mutations in neurodegenerative disorders.
Topics: Catalysis; Humans; Nucleoside-Phosphate Kinase; Phosphorylation; Protein Isoforms
PubMed: 34994281
DOI: 10.1080/15257770.2021.2023748 -
Progress in Histochemistry and... Nov 2016Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody... (Review)
Review
Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody specific for the protein in question. WB data is band(s) on a membrane while IHC result is a staining on a tissue section. Most human genes are known to produce multiple protein isoforms; in agreement with that, multiple bands are often found on the WB membrane. However, a common but unspoken practice in WB is to cut away the extra band(s) and present for publication only the band of interest, which implies to the readers that only one form of protein is expressed and thus the data interpretation is straightforward. Similarly, few IHC studies discuss whether the antibody used is isoform-specific and whether the positive staining is derived from only one isoform. Currently, there is no reliable technique to determine the isoform-specificity of an antibody, especially for IHC. Therefore, cutting away extra band(s) on the membrane usually is a form of misconduct in WB, and a positive staining in IHC only indicates the presence of protein product(s) of the to-be-interrogated gene, and not necessarily the presence of the isoform of interest. We suggest that data of WB and IHC involving only one antibody should not be published and that relevant reports should discuss whether there may be protein multiplicity and whether the antibody used is isoform-specific. Hopefully, techniques will soon emerge that allow determination of not only the presence of protein products of genes but also the isoforms expressed.
Topics: Alternative Splicing; Antibodies; Antibody Specificity; Bias; Blotting, Western; DNA-Binding Proteins; Humans; Immunohistochemistry; Mutant Chimeric Proteins; Nerve Tissue Proteins; Protein Isoforms; Scientific Misconduct
PubMed: 27908506
DOI: 10.1016/j.proghi.2016.11.001 -
International Journal of Molecular... Mar 2022Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin...
Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction.
Topics: Angiotensin-Converting Enzyme 2; COVID-19; Humans; Male; Protein Isoforms; SARS-CoV-2; Spermatozoa
PubMed: 35409054
DOI: 10.3390/ijms23073694 -
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 -
Journal of Proteome Research Jul 2022Alternative splicing can lead to distinct protein isoforms. These can have different functions in specific cells and tissues or in different developmental stages. In...
Alternative splicing can lead to distinct protein isoforms. These can have different functions in specific cells and tissues or in different developmental stages. In this study, we explored whether transcripts assembled from long read, nanopore-based, direct RNA-sequencing (RNA-seq) could improve the identification of protein isoforms in human K562 cells. By comparing with Illumina-based short read RNA-seq, we showed that a large proportion of Ensembl transcripts (5949/14,326) and genes expressing alternatively spliced transcripts (486/2981) identified with long direct reads were missed by short paired-end reads. By co-analyzing proteomic and transcriptomic data, we also showed that some peptides (826/35,976), proteins (262/3215), and protein isoforms arising from distinct transcript variants (574/1212) identified with isoform-specific peptides via custom long-read-based databases were missed in Illumina-derived databases. Finally, we generated unequivocal peptide evidence for a set of protein isoforms and showed that long read, direct RNA-seq allows the discovery of novel protein isoforms not already in reference databases or custom databases built from short read RNA-seq data. Our analysis highlights the benefits of long read RNA-seq data in the generation of reference databases to increase tandem mass spectrometry (MS/MS) identification of protein isoforms.
Topics: Alternative Splicing; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Humans; Peptides; Protein Isoforms; Proteomics; RNA; Sequence Analysis, RNA; Tandem Mass Spectrometry; Transcriptome
PubMed: 35612954
DOI: 10.1021/acs.jproteome.1c00968 -
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 -
BMB Reports Aug 2016Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau... (Review)
Review
Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413].
Topics: Humans; Mutation; Neurodegenerative Diseases; Protein Isoforms; RNA Splicing; tau Proteins
PubMed: 27222125
DOI: 10.5483/bmbrep.2016.49.8.084 -
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