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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 -
Journal of Cellular Biochemistry Apr 2023The human G protein coupled membrane receptor (GPR17), the sensor of brain damage, is identified as a biomarker for many neurological diseases. In human brain tissue,...
The human G protein coupled membrane receptor (GPR17), the sensor of brain damage, is identified as a biomarker for many neurological diseases. In human brain tissue, GPR17 exist in two isoforms, long and short. While cryo-electron microscopy technology has provided the structure of the long isoform of GPR17 with Gi complex, the structure of the short isoform and its activation mechanism remains unclear. Recently, we theoretically modeled the structure of the short isoform of GPR17 with Gi signaling protein and identified novel ligands. In the present work, we demonstrated the presence of two distinct ligand binding sites in the short isoform of GPR17. The molecular docking of GPR17 with endogenous (UDP) and synthetic ligands (T0510.3657, MDL29950) found the presence of two distinct binding pockets. Our observations revealed that endogenous ligand UDP can bind stronger in two different binding pockets as evidenced by glide and autodock vina scores, whereas the other two ligand's binding with GPR17 has less docking score. The analysis of receptor-UDP interactions shows complexes' stability in the lipid environment by 100 ns atomic molecular dynamics simulations. The amino acid residues VAL83, ARG87, and PHE111 constitute ligand binding site 1, whereas site 2 constitutes ASN67, ARG129, and LYS232. Root mean square fluctuation analysis showed the residues 83, 87, and 232 with higher fluctuations during molecular dynamics simulation in both binding pockets. Our findings imply that the residues of GPR17's two binding sites are crucial, and their interaction with UDP reveals the protein's hidden signaling and communication properties. Furthermore, this finding may assist in the development of targeted therapies for the treatment of neurological diseases.
Topics: Humans; Ligands; Molecular Docking Simulation; Cryoelectron Microscopy; Receptors, G-Protein-Coupled; Binding Sites; Uridine Diphosphate; Protein Isoforms
PubMed: 36791278
DOI: 10.1002/jcb.30388 -
Frontiers in Immunology 2023C-reactive protein (CRP) is an acute phase, predominantly hepatically synthesized protein, secreted in response to cytokine signaling at sites of tissue injury or... (Review)
Review
C-reactive protein (CRP) is an acute phase, predominantly hepatically synthesized protein, secreted in response to cytokine signaling at sites of tissue injury or infection with the physiological function of acute pro-inflammatory response. Historically, CRP has been classified as a mediator of the innate immune system, acting as a pattern recognition receptor for phosphocholine-containing ligands. For decades, CRP was envisioned as a single, non-glycosylated, multi-subunit protein arranged non-covalently in cyclic symmetry around a central void. Over the past few years, however, CRP has been shown to exist in at least three distinct isoforms: 1.) a pentamer of five identical globular subunits (pCRP), 2.) a modified monomer (mCRP) resulting from a conformational change when subunits are dissociated from the pentamer, and 3.) a transitional isoform where the pentamer remains intact but is partially changed to express mCRP structural characteristics (referred to as pCRP* or mCRP). The conversion of pCRP into mCRP can occur spontaneously and is observed under commonly used experimental conditions. In careful consideration of experimental design used in published reports of pro- and anti-inflammatory CRP bioactivities, we herein provide an interpretation of how distinctive CRP isoforms may have affected reported results. We argue that pro-inflammatory amplification mechanisms are consistent with the biofunction of mCRP, while weak anti-inflammatory mechanisms are consistent with pCRP. The interplay of each CRP isoform with specific immune cells (platelets, neutrophils, monocytes, endothelial cells, natural killer cells) and mechanisms of the innate immune system (complement), as well as differences in mCRP and pCRP ligand recognition and effector functions are discussed. This review will serve as a revised understanding of the structure-function relationship between CRP isoforms as related to inflammation and innate immunity mechanisms.
Topics: Humans; C-Reactive Protein; Endothelial Cells; Inflammation; Protein Isoforms; Immunity
PubMed: 37781355
DOI: 10.3389/fimmu.2023.1264383 -
Methods in Molecular Biology (Clifton,... 2022Conventional approaches to quantify alternative splicing are exon-centric and derive a ratio based on relative levels of the isoforms (or isoform groups) that include...
Conventional approaches to quantify alternative splicing are exon-centric and derive a ratio based on relative levels of the isoforms (or isoform groups) that include versus exclude a particular alternative RNA segment. The ratio measurement to study alternative splicing regulation can be confounded when alternative isoforms undergo differential RNA decay, for example, nonsense-mediated mRNA decay (NMD). Isoform-centric quantification is more informative for functional studies of alternative splicing, but challenges remain in distinguishing specific isoforms. Here, we provide a practical guide on addressing the specificity of isoform quantification and describe a simple sensitive method. Quantitative measurement of alternatively spliced RNA isoforms can be used to differentiate splicing regulation from transcriptional control and isoform-specific RNA decay regulation.
Topics: Alternative Splicing; Nonsense Mediated mRNA Decay; Protein Isoforms; RNA; RNA Isoforms; RNA, Messenger
PubMed: 35895259
DOI: 10.1007/978-1-0716-2521-7_5 -
Journal of Cell Science Feb 2022The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF)....
The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α-SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1-/- [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, SH2B1β and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1.
Topics: Adaptor Proteins, Signal Transducing; Animals; Brain-Derived Neurotrophic Factor; Cells, Cultured; Mice; Neurites; Neurons; Protein Isoforms
PubMed: 35019135
DOI: 10.1242/jcs.259179 -
Communications Biology Aug 2020While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in...
While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.
Topics: Histones; Protein Isoforms; Protein Processing, Post-Translational; Proteins; Single-Cell Analysis; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 32747637
DOI: 10.1038/s42003-020-01132-8 -
Biochemistry. Biokhimiia Oct 2023Bronchial asthma (BA) is a heterogeneous chronic inflammatory disease of the respiratory tract. Allergic (atopic) asthma is the most common (up to 80% of cases)... (Review)
Review
Bronchial asthma (BA) is a heterogeneous chronic inflammatory disease of the respiratory tract. Allergic (atopic) asthma is the most common (up to 80% of cases) phenotype developing through the Th2-dependent mechanisms involving cytokines: IL-4, IL-5, IL-9, and IL-13. The genes encoding Th2-cytokines have a mosaic structure (encode exons and introns). Therefore, several mature mRNA transcripts and protein isoforms can be derived from a single mRNA precursor through alternative splicing, and they may contribute to BA pathogenesis. Analysis of the published studies and databases revealed existence of the alternative mRNA transcripts for IL-4, IL-5, and IL-13. The alternative transcripts of IL-4 and IL-5 carry open reading frames and therefore can encode functional proteins. It was shown that not only alternative mRNA transcripts exist for IL-4, but alternative protein isoforms, as well. Natural protein isoform (IL-4δ2) lacking the part encoded by exon-2 was identified. Similarly, alternative mRNA transcript with deleted exon-2 (IL-5δ2) was also identified for IL-5. In this review, we summarize current knowledge about the identified alternative mRNA transcripts and protein isoforms of Th2-cytokinins, first of all IL-4 and IL-5. We have analyzed biological properties of the alternative variants of these cytokines, their possible role in the allergic asthma pathogenesis, and considered their diagnostic and therapeutic potential.
Topics: Humans; Cytokines; Alternative Splicing; Interleukin-4; Interleukin-5; Interleukin-13; Asthma; Protein Isoforms; RNA, Messenger; Th2 Cells
PubMed: 38105028
DOI: 10.1134/S0006297923100152 -
JCI Insight Jun 2023Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a...
Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector-mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X-KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector-treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector-mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.
Topics: Animals; Humans; Mice; Fear; Fragile X Mental Retardation Protein; Mice, Knockout; Protein Isoforms; Seizures
PubMed: 37288657
DOI: 10.1172/jci.insight.169650 -
Glia Jan 2024Tauopathies are a collection of neurodegenerative diseases characterized by the accumulation of pathogenic aggregates of the microtubule-associated protein tau. Despite...
Tauopathies are a collection of neurodegenerative diseases characterized by the accumulation of pathogenic aggregates of the microtubule-associated protein tau. Despite the prevalence and diversity of tau astrogliopathy in tauopathies, the interactions between astrocytes and tau in the brain, and the influence of neurodegenerative genetic risk factors like the apolipoprotein E4 (apoE4) isoform, are largely unknown. Here, we leveraged primary and immortalized astrocytes expressing humanized apoE isoforms to characterize the mechanisms by which astrocytes interact with and eliminate extracellular tau, and the influence of apoE genotype on these processes. Our work indicates that astrocytes rapidly internalize, process, and release tau via an exosomal secretory mechanism under physiological conditions. However, we found that apoE4 disrupted these processes in comparison to apoE3, resulting in an astrocytic phenotype prone to intracellular tau accumulation. Furthermore, exposure to repetitive mild traumatic brain injuries exacerbated the apoE4-induced impairments in tau processing and elimination by astrocytes in apoE4 targeted-replacement mice. The diminished ability of apoE4 astrocytes to eliminate extracellular tau can lead to an accumulation of pathogenic tau, which induces mitochondrial dysfunction, as demonstrated by our studies. In total, our findings suggest that the apoE4 isoform lowers the threshold of astrocytic resilience to pathogenic tau, rendering them susceptible to bioenergetic deficits in the early stages of neurodegenerative diseases such as traumatic brain injury, potentially contributing to neurological decline.
Topics: Mice; Animals; Apolipoprotein E4; Astrocytes; Mice, Transgenic; Apolipoproteins E; Tauopathies; Neurodegenerative Diseases; Protein Isoforms
PubMed: 37668005
DOI: 10.1002/glia.24469