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Nature Reviews. Cancer Mar 2023Dysregulated RNA splicing is a molecular feature that characterizes almost all tumour types. Cancer-associated splicing alterations arise from both recurrent mutations... (Review)
Review
Dysregulated RNA splicing is a molecular feature that characterizes almost all tumour types. Cancer-associated splicing alterations arise from both recurrent mutations and altered expression of trans-acting factors governing splicing catalysis and regulation. Cancer-associated splicing dysregulation can promote tumorigenesis via diverse mechanisms, contributing to increased cell proliferation, decreased apoptosis, enhanced migration and metastatic potential, resistance to chemotherapy and evasion of immune surveillance. Recent studies have identified specific cancer-associated isoforms that play critical roles in cancer cell transformation and growth and demonstrated the therapeutic benefits of correcting or otherwise antagonizing such cancer-associated mRNA isoforms. Clinical-grade small molecules that modulate or inhibit RNA splicing have similarly been developed as promising anticancer therapeutics. Here, we review splicing alterations characteristic of cancer cell transcriptomes, dysregulated splicing's contributions to tumour initiation and progression, and existing and emerging approaches for targeting splicing for cancer therapy. Finally, we discuss the outstanding questions and challenges that must be addressed to translate these findings into the clinic.
Topics: Humans; Alternative Splicing; RNA Splicing; Neoplasms; Protein Isoforms; Carcinogenesis; Cell Transformation, Neoplastic
PubMed: 36627445
DOI: 10.1038/s41568-022-00541-7 -
Journal of Medical Genetics Oct 2005Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA... (Review)
Review
Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited.
Topics: Alternative Splicing; Base Sequence; Binding Sites; Exons; Genetic Predisposition to Disease; Humans; Introns; Models, Genetic; Molecular Sequence Data; Mutation; Protein Isoforms; RNA Precursors; RNA Splicing; RNA, Messenger
PubMed: 16199547
DOI: 10.1136/jmg.2004.029538 -
Nature Cancer May 2022High-throughput sequencing and functional characterization of the cancer transcriptome have uncovered cancer-specific dysregulation of RNA splicing across a variety of... (Review)
Review
High-throughput sequencing and functional characterization of the cancer transcriptome have uncovered cancer-specific dysregulation of RNA splicing across a variety of cancers. Alterations in the cancer genome and dysregulation of RNA splicing factors lead to missplicing, splicing alteration-dependent gene expression and, in some cases, generation of novel splicing-derived proteins. Here, we review recent advances in our understanding of aberrant splicing in cancer pathogenesis and present strategies to harness cancer-specific aberrant splicing for therapeutic intent.
Topics: Humans; Neoplasms; RNA Splicing; RNA Splicing Factors
PubMed: 35624337
DOI: 10.1038/s43018-022-00384-z -
American Journal of Human Genetics Jul 2023The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) framework for classifying variants uses six evidence categories...
The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) framework for classifying variants uses six evidence categories related to the splicing potential of variants: PVS1, PS3, PP3, BS3, BP4, and BP7. However, the lack of guidance on how to apply such codes has contributed to variation in the specifications developed by different Clinical Genome Resource (ClinGen) Variant Curation Expert Panels. The ClinGen Sequence Variant Interpretation Splicing Subgroup was established to refine recommendations for applying ACMG/AMP codes relating to splicing data and computational predictions. We utilized empirically derived splicing evidence to (1) determine the evidence weighting of splicing-related data and appropriate criteria code selection for general use, (2) outline a process for integrating splicing-related considerations when developing a gene-specific PVS1 decision tree, and (3) exemplify methodology to calibrate splice prediction tools. We propose repurposing the PVS1_Strength code to capture splicing assay data that provide experimental evidence for variants resulting in RNA transcript(s) with loss of function. Conversely, BP7 may be used to capture RNA results demonstrating no splicing impact for intronic and synonymous variants. We propose that the PS3/BS3 codes are applied only for well-established assays that measure functional impact not directly captured by RNA-splicing assays. We recommend the application of PS1 based on similarity of predicted RNA-splicing effects for a variant under assessment in comparison with a known pathogenic variant. The recommendations and approaches for consideration and evaluation of RNA-assay evidence described aim to help standardize variant pathogenicity classification processes when interpreting splicing-based evidence.
Topics: Humans; United States; Genetic Variation; Genome, Human; Genomics; Alleles; RNA Splicing; Genetic Testing
PubMed: 37352859
DOI: 10.1016/j.ajhg.2023.06.002 -
Signal Transduction and Targeted Therapy Feb 2021The abnormal regulation of alternative splicing is usually accompanied by the occurrence and development of tumors, which would produce multiple different isoforms and...
The abnormal regulation of alternative splicing is usually accompanied by the occurrence and development of tumors, which would produce multiple different isoforms and diversify protein expression. The aim of the present study was to conduct a systematic review in order to describe the regulatory mechanisms of alternative splicing, as well as its functions in tumor cells, from proliferation and apoptosis to invasion and metastasis, and from angiogenesis to metabolism. The abnormal splicing events contributed to tumor progression as oncogenic drivers and/or bystander factors. The alterations in splicing factors detected in tumors and other mis-splicing events (i.e., long non-coding and circular RNAs) in tumorigenesis were also included. The findings of recent therapeutic approaches targeting splicing catalysis and splicing regulatory proteins to modulate pathogenically spliced events (including tumor-specific neo-antigens for cancer immunotherapy) were introduced. The emerging RNA-based strategies for the treatment of cancer with abnormally alternative splicing isoforms were also discussed. However, further studies are still required to address the association between alternative splicing and cancer in more detail.
Topics: Alternative Splicing; Carcinogenesis; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; RNA Splicing; RNA Splicing Factors; RNA, Circular
PubMed: 33623018
DOI: 10.1038/s41392-021-00486-7 -
Methods (San Diego, Calif.) Dec 2021Circular RNAs (circRNAs) are a novel class of RNAs distinguished by their single-stranded, covalently-closed topology. Although initially perceived as rare byproducts of... (Review)
Review
Circular RNAs (circRNAs) are a novel class of RNAs distinguished by their single-stranded, covalently-closed topology. Although initially perceived as rare byproducts of aberrant splicing, circRNAs are now recognized as ubiquitously expressed and functionally significant. These discoveries have led to a growing need for ways to model circRNAs in living cells to advance our understanding of their biogenesis, regulation, and function, and to adopt them as new technologies for application within research and medicine. In this review, we provide an updated summary of approaches used to produce circRNAs in vitro and in vivo, the latter of which has grown considerably in recent years. Given increased interest in the unique functions carried out by individual circRNAs, we further dedicate a section on how to customize synthesized circRNAs for specific biological roles. We focus on the most common applications, including designing circRNAs for protein delivery, to target miRNAs and proteins, to act as fluorescent reporters, and to modulate cellular immunity.
Topics: MicroRNAs; Proteins; RNA Splicing; RNA, Circular
PubMed: 33662562
DOI: 10.1016/j.ymeth.2021.02.020 -
Nucleic Acids Research Mar 2023Alternative splicing is an important source of heterogeneity underlying gene expression between individual cells but remains an understudied area due to the paucity of...
Alternative splicing is an important source of heterogeneity underlying gene expression between individual cells but remains an understudied area due to the paucity of computational tools to analyze splicing dynamics at single-cell resolution. Here, we present MARVEL, a comprehensive R package for single-cell splicing analysis applicable to RNA sequencing generated from the plate- and droplet-based methods. We performed extensive benchmarking of MARVEL against available tools and demonstrated its utility by analyzing multiple publicly available datasets in diverse cell types, including in disease. MARVEL enables systematic and integrated splicing and gene expression analysis of single cells to characterize the splicing landscape and reveal biological insights.
Topics: Alternative Splicing; Software; Computational Biology; RNA Splicing; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 36631981
DOI: 10.1093/nar/gkac1260 -
Cell May 2021The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the...
The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.
Topics: Exons; Humans; Introns; RNA Precursors; RNA Splice Sites; RNA Splicing; RNA, Messenger; Spliceosomes; Transcription, Genetic; Transcriptome
PubMed: 33979654
DOI: 10.1016/j.cell.2021.04.012 -
Nucleic Acids Research Aug 2022SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in...
SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence and concentration dependence supported by in vitro experiments. Live-cell imaging shows that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.
Topics: Alternative Splicing; Exons; HEK293 Cells; Humans; Introns; Protein Isoforms; RNA Splicing; RNA-Binding Proteins
PubMed: 35929045
DOI: 10.1093/nar/gkac669 -
PLoS Biology May 2023Leukemogenesis is proposed to be a multistep process by which normal hematopoietic stem and progenitor cells are transformed into full-blown leukemic cells, the details...
Leukemogenesis is proposed to be a multistep process by which normal hematopoietic stem and progenitor cells are transformed into full-blown leukemic cells, the details of which are not fully understood. Here, we performed serial single-cell transcriptome analyses of preleukemic and leukemic cells (PLCs) and constructed the cellular and molecular transformation trajectory in a Myc-driven acute myeloid leukemia (AML) model in mice, which represented the transformation course in patients. We found that the Myc targets were gradually up-regulated along the trajectory. Among them were splicing factors, which showed stage-specific prognosis for AML patients. Furthermore, we dissected the detailed gene network of a tipping point for hematopoietic stem and progenitor cells (HSPCs) to generate initiating PLCs, which was characterized by dramatically increased splicing factors and unusual RNA velocity. In the late stage, PLCs acquired explosive heterogeneity through RNA alternative splicing. Among them, the Hsp90aa1hi subpopulation was conserved in both human and mouse AML and associated with poor prognosis. Exon 4 skipping of Tmem134 was identified in these cells. While the exon skipping product Tmem134β promoted the cell cycle, full-length Tmem134α delayed tumorigenesis. Our study emphasized the critical roles of RNA splicing in the full process of leukemogenesis.
Topics: Humans; Animals; Mice; Single-Cell Gene Expression Analysis; Leukemia, Myeloid, Acute; RNA Splicing; RNA; RNA Splicing Factors; Transcriptome
PubMed: 37130348
DOI: 10.1371/journal.pbio.3002088