Review

Authors: Robert K Bradley, Olga Anczuków

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

Authors: Logan C Walker, Miguel de la Hoya, George A R Wiggins...

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

Review

Authors: D Baralle, M Baralle

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

Review

Authors: Robert F Stanley, Omar Abdel-Wahab

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

Review

Authors: Hossein Shenasa, David L Bentley

Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.

Topics: RNA Precursors; Transcription, Genetic; RNA Splicing; Spliceosomes; Introns

PubMed: 37236814
DOI: 10.1016/j.tig.2023.04.008

Authors: Yuanjiao Zhang, Jinjun Qian, Chunyan Gu...

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

Review

Authors: Jimena Giudice, Hao Jiang

Biomolecular condensates, sometimes also known as membraneless organelles (MLOs), can form through weak multivalent intermolecular interactions of proteins and nucleic acids, a process often associated with liquid-liquid phase separation. Biomolecular condensates are emerging as sites and regulatory platforms of vital cellular functions, including transcription and RNA processing. In the first part of this Review, we comprehensively discuss how alternative splicing regulates the formation and properties of condensates, and conversely the roles of biomolecular condensates in splicing regulation. In the second part, we focus on the spatial connection between splicing regulation and nuclear MLOs such as transcriptional condensates, splicing condensates and nuclear speckles. We then discuss key studies showing how splicing regulation through biomolecular condensates is implicated in human pathologies such as neurodegenerative diseases, different types of cancer, developmental disorders and cardiomyopathies, and conclude with a discussion of outstanding questions pertaining to the roles of condensates and MLOs in splicing regulation and how to experimentally study them.

Topics: Humans; Biomolecular Condensates; Organelles; Animals; Alternative Splicing; RNA Splicing; Cell Nucleus

PubMed: 38773325
DOI: 10.1038/s41580-024-00739-7

Review

Authors: Prisca Obi, Y Grace Chen

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

Authors: Wei Xiong Wen, Adam J Mead, Supat Thongjuea...

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

Authors: Satoru Shinriki, Mayumi Hirayama, Akiko Nagamachi...

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

Topics: Cell Line; RNA Splicing; RNA Splice Sites; Mutation; DNA Replication

PubMed: 36229594
DOI: 10.1038/s41375-022-01708-9