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Pharmacological Research Dec 2023Sorafenib, a multi-targeted tyrosine kinase inhibitor, is a first-line treatment for advanced solid tumors, but it induces many adverse cardiovascular events, including...
Sorafenib, a multi-targeted tyrosine kinase inhibitor, is a first-line treatment for advanced solid tumors, but it induces many adverse cardiovascular events, including myocardial infarction and heart failure. These cardiac defects can be mediated by alternative splicing of genes critical for heart function. Whether alternative splicing plays a role in sorafenib-induced cardiotoxicity remains unclear. Transcriptome of rat hearts or human cardiomyocytes treated with sorafenib was analyzed and validated to define alternatively spliced genes and their impact on cardiotoxicity. In rats, sorafenib caused severe cardiotoxicity with decreased left ventricular systolic pressure, elongated sarcomere, enlarged mitochondria and decreased ATP. This was associated with alternative splicing of hundreds of genes in the hearts, many of which were targets of a cardiac specific splicing factor, RBM20. Sorafenib inhibited RBM20 expression in both rat hearts and human cardiomyocytes. The splicing of RBM20's targets, SLC25A3 and FHOD3, was altered into fetal isoforms with decreased function. Upregulation of RBM20 during sorafenib treatment reversed the pathogenic splicing of SLC25A3 and FHOD3, and enhanced the phosphate transport into mitochondria by SLC25A3, ATP synthesis and cell survival.We envision this regulation may happen in many drug-induced cardiotoxicity, and represent a potential druggable pathway for mitigating sorafenib-induced cardiotoxicity.
Topics: Rats; Animals; Humans; Alternative Splicing; Sorafenib; Cardiotoxicity; Sarcomeres; Genes, Mitochondrial; RNA-Binding Proteins; Myocytes, Cardiac; Adenosine Triphosphate; Formins
PubMed: 38006979
DOI: 10.1016/j.phrs.2023.107017 -
BioEssays : News and Reviews in... Sep 2023Fold-switching proteins, which remodel their secondary and tertiary structures in response to cellular stimuli, suggest a new view of protein fold space. For decades,...
Fold-switching proteins, which remodel their secondary and tertiary structures in response to cellular stimuli, suggest a new view of protein fold space. For decades, experimental evidence has indicated that protein fold space is discrete: dissimilar folds are encoded by dissimilar amino acid sequences. Challenging this assumption, fold-switching proteins interconnect discrete groups of dissimilar protein folds, making protein fold space fluid. Three recent observations support the concept of fluid fold space: (1) some amino acid sequences interconvert between folds with distinct secondary structures, (2) some naturally occurring sequences have switched folds by stepwise mutation, and (3) fold switching is evolutionarily selected and likely confers advantage. These observations indicate that minor amino acid sequence modifications can transform protein structure and function. Consequently, proteomic structural and functional diversity may be expanded by alternative splicing, small nucleotide polymorphisms, post-translational modifications, and modified translation rates.
Topics: Protein Folding; Proteomics; Models, Molecular; Proteins; Amino Acid Sequence
PubMed: 37431685
DOI: 10.1002/bies.202300057 -
PLoS Genetics Sep 2023Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during...
Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during neuronal morphogenesis is not fully understood. Here, we show that LIM homeodomain protein MEC-3, the key TF for C. elegans PVD dendrite morphogenesis, is regulated by both alternative splicing and an E3 ubiquitin ligase. The mec-3 gene generates several transcripts by alternative splicing. We find that mbl-1, the orthologue of the muscular dystrophy disease gene muscleblind-like (MBNL), is required for PVD dendrite arbor formation. Our data suggest mbl-1 regulates the alternative splicing of mec-3 to produce its long isoform. Deleting the long isoform of mec-3(deExon2) causes reduction of dendrite complexity. Through a genetic modifier screen, we find that mutation in the E3 ubiquitin ligase EEL-1 suppresses mbl-1 phenotype. eel-1 mutants also suppress mec-3(deExon2) mutant but not the mec-3 null phenotype. Loss of EEL-1 alone leads to excessive dendrite branches. Together, these results indicate that MEC-3 is fine-tuned by alternative splicing and the ubiquitin system to produce the optimal level of dendrite branches.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Dendrites; Eels; RNA Splicing; Ubiquitin-Protein Ligases
PubMed: 37729192
DOI: 10.1371/journal.pgen.1010941 -
The International Journal of... Sep 2023The faithful splicing of pre-mRNA is critical for accurate gene expression. Dysregulation of pre-mRNA splicing has been associated with several human diseases including... (Review)
Review
The faithful splicing of pre-mRNA is critical for accurate gene expression. Dysregulation of pre-mRNA splicing has been associated with several human diseases including cancer. The ubiquitin-like protein Hub1/UBL5 binds to the substrates non-covalently and promotes pre-mRNA splicing. Additionally, UBL5 promotes the common fragile sites stability and the Fanconi anemia pathway of DNA damage repair. These functions strongly suggests that UBL5 could potentially be implicated in cancer. Therefore, we analyzed the UBL5 expression in TCGA tumor sample datasets and observed the differences between tumor and normal tissues among different tumor subtypes. We have noticed the alteration frequency of UBL5 in TCGA tumor samples. Altogether, this review summarizes the UBL5 functions and discusses its putative role in tumorigenesis.
Topics: Humans; RNA Precursors; RNA Splicing; Ubiquitins
PubMed: 37453225
DOI: 10.1016/j.biocel.2023.106445 -
Molecular Therapy. Nucleic Acids Sep 2023Fukutin () c.647+2084G>T creates a pseudo-exon with a premature stop codon, which causes Fukuyama congenital muscular dystrophy (FCMD). We aimed to ameliorate aberrant...
Fukutin () c.647+2084G>T creates a pseudo-exon with a premature stop codon, which causes Fukuyama congenital muscular dystrophy (FCMD). We aimed to ameliorate aberrant splicing of caused by this variant. We screened compounds focusing on splicing regulation using the c.647+2084G>T splicing reporter and discovered that the branchpoint, which is essential for splicing reactions, could be a potential therapeutic target. To confirm the effectiveness of branchpoints as targets for exon skipping, we designed branchpoint-targeted antisense oligonucleotides (BP-AONs). This restored normal mRNA and protein production in FCMD patient myotubes. We identified a functional BP by detecting splicing intermediates and creating BP mutations in the reporter gene; this BP was non-redundant and sufficiently blocked by BP-AONs. Next, a BP-AON was designed for a different FCMD-causing variant, which induces pathogenic exon trapping by a common SINE-VNTR-Alu-type retrotransposon. Notably, this BP-AON also restored normal mRNA and protein production in FCMD patient myotubes. Our findings suggest that BPs could be potential targets in exon-skipping therapeutic strategies for genetic disorders.
PubMed: 37547287
DOI: 10.1016/j.omtn.2023.07.011 -
The Journal of Clinical Investigation Sep 2023RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation...
RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrated that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segregation, altered spindle architecture, and aneuploidy, which could be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlay augmentation of cen-R-loop, as overexpression of the normal isoform, but not the altered form, mitigated mitotic stress in SF3B1-mutant cells. Our study identifies a critical role of splice variants in linking RNA splicing dysregulation and CIN and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.
Topics: Humans; Leukemia, Lymphocytic, Chronic, B-Cell; R-Loop Structures; Phosphoproteins; Mutation; RNA Splicing Factors; Ataxia Telangiectasia Mutated Proteins
PubMed: 37463047
DOI: 10.1172/JCI163325 -
Frontiers in Plant Science 2023For the past two decades, the study of alternative splicing (AS) and its involvement in plant development and stress response has grown in popularity. Only recently... (Review)
Review
For the past two decades, the study of alternative splicing (AS) and its involvement in plant development and stress response has grown in popularity. Only recently however, has the focus shifted to the study of how AS regulation (or lack-thereof) affects downstream mRNA and protein landscapes and how these AS regulatory events impact plant development and stress tolerance. In humans, protein phosphorylation represents one of the predominant mechanisms by which AS is regulated and thus the protein kinases governing these phosphorylation events are of interest for further study. Large-scale phosphoproteomic studies in plants have consistently found that RNA splicing-related proteins are extensively phosphorylated, however, the signaling pathways involved in AS regulation have not been resolved. In this mini-review, we summarize our current knowledge of the three major splicing-related protein kinase families in plants that are suggested to mediate AS phospho-regulation and draw comparisons to their metazoan orthologs. We also summarize and contextualize the phosphorylation events identified as occurring on splicing-related protein families to illustrate the high degree to which splicing-related proteins are modified, placing a new focus on elucidating the impacts of AS at the protein and PTM-level.
PubMed: 37780493
DOI: 10.3389/fpls.2023.1249057 -
Molecular Neurodegeneration Sep 2023ApoE4, the most significant genetic risk factor for late-onset Alzheimer's disease (AD), sequesters a pro-synaptogenic Reelin receptor, Apoer2, in the endosomal...
BACKGROUND
ApoE4, the most significant genetic risk factor for late-onset Alzheimer's disease (AD), sequesters a pro-synaptogenic Reelin receptor, Apoer2, in the endosomal compartment and prevents its normal recycling. In the adult brain, Reelin potentiates excitatory synapses and thereby protects against amyloid-β toxicity. Recently, a gain-of-function mutation in Reelin that is protective against early-onset AD has been described. Alternative splicing of the Apoer2 intracellular domain (Apoer2-ICD) regulates Apoer2 signaling. Splicing of juxtamembraneous exon 16 alters the γ-secretase mediated release of the Apoer2-ICD as well as synapse number and LTP, and inclusion of exon 19 ameliorates behavioral deficits in an AD mouse model. The Apoer2-ICD has also been shown to alter transcription of synaptic genes. However, the role of Apoer2-ICD release upon transcriptional regulation and its role in AD pathogenesis is unknown.
METHODS
To assess in vivo mRNA-primed ribosomes specifically in hippocampi transduced with Apoer2-ICD splice variants, we crossed wild-type, cKO, and Apoer2 cleavage-resistant mice to a Cre-inducible translating ribosome affinity purification (TRAP) model. This allowed us to perform RNA-Seq on ribosome-loaded mRNA harvested specifically from hippocampal cells transduced with Apoer2-ICDs.
RESULTS
Across all conditions, we observed ~4,700 altered translating transcripts, several of which comprise key synaptic components such as extracellular matrix and focal adhesions with concomitant perturbation of critical signaling cascades, energy metabolism, translation, and apoptosis. We further demonstrated the ability of the Apoer2-ICD to rescue many of these altered transcripts, underscoring the importance of Apoer2 splicing in synaptic homeostasis. A variety of these altered genes have been implicated in AD, demonstrating how dysregulated Apoer2 splicing may contribute to neurodegeneration.
CONCLUSIONS
Our findings demonstrate how alternative splicing of the APOE and Reelin receptor Apoer2 and release of the Apoer2-ICD regulates numerous translating transcripts in mouse hippocampi in vivo. These transcripts comprise a wide range of functions, and alterations in these transcripts suggest a mechanistic basis for the synaptic deficits seen in Apoer2 mutant mice and AD patients. Our findings, together with the recently reported AD-protective effects of a Reelin gain-of-function mutation in the presence of an early-onset AD mutation in Presenilin-1, implicate the Reelin/Apoer2 pathway as a target for AD therapeutics.
Topics: Animals; Mice; Alternative Splicing; Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; RNA Splicing
PubMed: 37726747
DOI: 10.1186/s13024-023-00652-1 -
Journal of the American Chemical Society Oct 2023R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells and are enriched in promoter regions of genes. By...
R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) data sets, we found that many splicing factors, including U2AF1 whose recognition of the 3' splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We also revealed that U2AF1 binds directly to R-loops and DNA G4 structures at a low-nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1's interaction with 3' splicing site and leads to augmented distribution of RNA polymerase II (RNAPII) to promoters over gene bodies, thereby modulating cotranscriptional pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interactions of U2AF1 with these nucleic acid structures, and established a biochemical rationale for U2AF1's occupancy in gene promoters. We also unveiled that interaction with R-loops promotes U2AF1's phase separation, and our work suggests that U2AF1 modulates pre-mRNA splicing by regulating RNAPII's partition in transcription initiation versus elongation.
Topics: Humans; Splicing Factor U2AF; R-Loop Structures; RNA Precursors; RNA Splicing; RNA-Binding Proteins; DNA; RNA; Promoter Regions, Genetic
PubMed: 37733759
DOI: 10.1021/jacs.3c08204 -
Cell Death & Disease Oct 2023Abnormal alternative splicing (AS) caused by alterations in spliceosomal factors is implicated in cancers. Standard models posit that splice site selection is mainly...
Abnormal alternative splicing (AS) caused by alterations in spliceosomal factors is implicated in cancers. Standard models posit that splice site selection is mainly determined by early spliceosomal U1 and U2 snRNPs. Whether and how other mid/late-acting spliceosome components such as USP39 modulate tumorigenic splice site choice remains largely elusive. We observed that hepatocyte-specific overexpression of USP39 promoted hepatocarcinogenesis and potently regulated splice site selection in transgenic mice. In human liver cancer cells, USP39 promoted tumor proliferation in a spliceosome-dependent manner. USP39 depletion deregulated hundreds of AS events, including the oncogenic splice-switching of KANK2. Mechanistically, we developed a novel RBP-motif enrichment analysis and found that USP39 modulated exon inclusion/exclusion by interacting with SRSF6/HNRNPC in both humans and mice. Our data represented a paradigm for the control of splice site selection by mid/late-acting spliceosome proteins and their interacting RBPs. USP39 and possibly other mid/late-acting spliceosome proteins may represent potential prognostic biomarkers and targets for cancer therapy.
Topics: Humans; Mice; Animals; Alternative Splicing; Carcinoma, Hepatocellular; Liver Neoplasms; RNA Splicing; Carcinogenesis; Serine-Arginine Splicing Factors; Phosphoproteins; Heterogeneous-Nuclear Ribonucleoprotein Group C; Ubiquitin-Specific Proteases
PubMed: 37821439
DOI: 10.1038/s41419-023-06210-3