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Acta Pharmaceutica Sinica. B Aug 2023Serine/arginine-rich splicing factors (SRSFs) refer to twelve RNA-binding proteins which regulate splice site recognition and spliceosome assembly during precursor... (Review)
Review
Serine/arginine-rich splicing factors (SRSFs) refer to twelve RNA-binding proteins which regulate splice site recognition and spliceosome assembly during precursor messenger RNA splicing. SRSFs also participate in other RNA metabolic events, such as transcription, translation and nonsense-mediated decay, during their shuttling between nucleus and cytoplasm, making them indispensable for genome diversity and cellular activity. Of note, aberrant SRSF expression and/or mutations elicit fallacies in gene splicing, leading to the generation of pathogenic gene and protein isoforms, which highlights the therapeutic potential of targeting SRSF to treat diseases. In this review, we updated current understanding of SRSF structures and functions in RNA metabolism. Next, we analyzed SRSF-induced aberrant gene expression and their pathogenic outcomes in cancers and non-tumor diseases. The development of some well-characterized SRSF inhibitors was discussed in detail. We hope this review will contribute to future studies of SRSF functions and drug development targeting SRSFs.
PubMed: 37655328
DOI: 10.1016/j.apsb.2023.05.022 -
Nature Feb 2024The assembly and specification of synapses in the brain is incompletely understood. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion...
The assembly and specification of synapses in the brain is incompletely understood. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gα signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gα or Gα. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gα- to a Gα-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gα signalling by LPHN3 splice variants mediates synapse formation. Notably, Gα-coupled, but not Gα-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gα-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gα signalling and clustered phase separation of postsynaptic protein scaffolds.
Topics: Animals; Mice; Alternative Splicing; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Protein alpha Subunits, Gs; Ligands; Receptors, G-Protein-Coupled; Receptors, Peptide; Synapses; Signal Transduction
PubMed: 38233523
DOI: 10.1038/s41586-023-06913-9 -
Genes & Development Dec 2023RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA... (Review)
Review
RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA complexes remains poorly understood. In this issue of , Zhang and colleagues (pp. 968-983) combine biochemical experiments with AI-based structure prediction methods to generate a model for the interaction between SF3B1, a core splicing factor essential for the recognition of the intron branchpoint, and SUGP1, a protein that bridges SF3B1 with the helicase DHX15. Interaction with SF3B1 exposes the G-patch domain of SUGP1, facilitating binding to and activation of DHX15. The model can explain the activation of cryptic 3' splice sites induced by mutations in SF3B1 or SUGP1 frequently found in cancer.
Topics: RNA Splicing; Spliceosomes; RNA Splicing Factors; RNA Splice Sites; RNA Precursors; Artificial Intelligence; Mutation; Phosphoproteins
PubMed: 38092520
DOI: 10.1101/gad.351373.123 -
Frontiers in Microbiology 2023Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context,... (Review)
Review
Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.
PubMed: 38029209
DOI: 10.3389/fmicb.2023.1305848 -
Journal of Hepatology Apr 2024Intrahepatic cholangiocarcinoma (iCCA) is a fatal malignancy of the biliary system. The lack of a detailed understanding of oncogenic signaling or global gene expression...
BACKGROUND & AIMS
Intrahepatic cholangiocarcinoma (iCCA) is a fatal malignancy of the biliary system. The lack of a detailed understanding of oncogenic signaling or global gene expression alterations has impeded clinical iCCA diagnosis and therapy. The role of protein lactylation, a newly unraveled post-translational modification that orchestrates gene expression, remains largely elusive in the pathogenesis of iCCA.
METHODS
Proteomics analysis of clinical iCCA specimens and adjacent tissues was performed to screen for proteins aberrantly lactylated in iCCA. Mass spectrometry, macromolecule interaction and cell behavioral studies were employed to identify the specific lactylation sites on the candidate protein(s) and to decipher the downstream mechanisms responsible for iCCA development, which were subsequently validated using a xenograft tumor model and clinical samples.
RESULTS
Nucleolin (NCL), the most abundant RNA-binding protein in the nucleolus, was identified as a functional lactylation target that correlates with iCCA occurrence and progression. NCL was lactylated predominantly at lysine 477 by the acyltransferase P300 in response to a hyperactivity of glycolysis, and promoted the proliferation and invasion of iCCA cells. Mechanistically, lactylated NCL bound to the primary transcript of MAP kinase-activating death domain protein (MADD) and warranted an efficient translation of MADD by circumventing alternative splicing that generates a premature termination codon. NCL lactylation, MADD and subsequent ERK activation promoted xenograft tumor growth, and were found to associate with the overall survival of iCCA patients.
CONCLUSION
NCL is lactylated to upregulate MADD through an RNA splicing-dependent mechanism, which potentiates iCCA pathogenesis via the MAPK pathway. Our findings reveal a novel link between metabolic reprogramming and canonical tumor-initiating events, and provide biomarkers that can be potentially used for prognostic evaluation or targeted treatment of iCCA.
IMPACT AND IMPLICATION
Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive liver malignancy with largely uncharacterized pathogenetic mechanisms. Herein, we demonstrated that glycolysis promotes P300-catalyzed lactylation of NCL, which upregulates MAP kinase-activating death domain protein (MADD) through precise mRNA splicing, and activates ERK signaling to drive iCCA development. These findings unravel a novel link between metabolic rewiring and canonical oncogenic pathways, and provide new biomarkers for prognostic assessment and targeting of clinical iCCA.
PubMed: 38679071
DOI: 10.1016/j.jhep.2024.04.010 -
Molecular Cancer Jan 2024Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a...
BACKGROUND
Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown.
METHODS
High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis.
RESULTS
In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC.
CONCLUSIONS
Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.
Topics: Humans; Carcinoma, Renal Cell; Alternative Splicing; RNA, Circular; MicroRNAs; Kidney Neoplasms; Heterogeneous-Nuclear Ribonucleoproteins; Polypyrimidine Tract-Binding Protein; Cytochrome P-450 CYP1B1; Heterogeneous-Nuclear Ribonucleoprotein Group C
PubMed: 38184608
DOI: 10.1186/s12943-023-01912-w -
BioRxiv : the Preprint Server For... Dec 2023Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated...
Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.
PubMed: 38187674
DOI: 10.1101/2023.12.18.572199 -
Nature Communications Oct 2023Alternative splicing generates functional diversity in isoforms, impacting immune response to infection. Here, we evaluate the causal role of alternative splicing in...
Alternative splicing generates functional diversity in isoforms, impacting immune response to infection. Here, we evaluate the causal role of alternative splicing in COVID-19 severity and susceptibility by applying two-sample Mendelian randomization to cis-splicing quantitative trait loci and the results from COVID-19 Host Genetics Initiative. We identify that alternative splicing in lung, rather than total expression of OAS1, ATP11A, DPP9 and NPNT, is associated with COVID-19 severity. MUC1 and PMF1 splicing is associated with COVID-19 susceptibility. Colocalization analyses support a shared genetic mechanism between COVID-19 severity with idiopathic pulmonary fibrosis at the ATP11A and DPP9 loci, and with chronic obstructive lung diseases at the NPNT locus. Last, we show that ATP11A, DPP9, NPNT, and MUC1 are highly expressed in lung alveolar epithelial cells, both in COVID-19 uninfected and infected samples. These findings clarify the importance of alternative splicing in lung for COVID-19 and respiratory diseases, providing isoform-based targets for drug discovery.
Topics: Humans; Alternative Splicing; Genetic Predisposition to Disease; COVID-19; Lung; Pulmonary Disease, Chronic Obstructive; Protein Isoforms; Respiration Disorders; Genome-Wide Association Study
PubMed: 37794074
DOI: 10.1038/s41467-023-41912-4 -
Wiley Interdisciplinary Reviews. RNA 2023The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation.... (Review)
Review
The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Topics: Child; Humans; RNA Splicing; RNA; Alternative Splicing; RNA-Binding Proteins
PubMed: 37042074
DOI: 10.1002/wrna.1788 -
Annual Review of Biomedical Data Science Aug 2023Alternative splicing is pivotal to the regulation of gene expression and protein diversity in eukaryotic cells. The detection of alternative splicing events requires... (Review)
Review
Alternative splicing is pivotal to the regulation of gene expression and protein diversity in eukaryotic cells. The detection of alternative splicing events requires specific omics technologies. Although short-read RNA sequencing has successfully supported a plethora of investigations on alternative splicing, the emerging technologies of long-read RNA sequencing and top-down mass spectrometry open new opportunities to identify alternative splicing and protein isoforms with less ambiguity. Here, we summarize improvements in short-read RNA sequencing for alternative splicing analysis, including percent splicing index estimation and differential analysis. We also review the computational methods used in top-down proteomics analysis regarding proteoform identification, including the construction of databases of protein isoforms and statistical analyses of search results. While many improvements in sequencing and computational methods will result from emerging technologies, there should be future endeavors to increase the effectiveness, integration, and proteome coverage of alternative splicing events.
Topics: Proteomics; Transcriptome; Protein Isoforms; Alternative Splicing; RNA Splicing
PubMed: 37561601
DOI: 10.1146/annurev-biodatasci-020722-044021