Rare and private spliceosomal gene mutations drive partial, complete, and dual phenocopies of hotspot alterations

Genes encoding the RNA splicing factors SF3B1, SRSF2, and U2AF1 are subject to frequent missense mutations in clonal hematopoiesis and diverse neoplastic diseases. Most "spliceosomal" mutations affect specific hotspot residues, resulting in splicing changes that promote disease pathophysiology. However, a subset of patients carry spliceosomal mutations that affect non-hotspot residues, whose potential functional contributions to disease are unstudied. Here, we undertook a systematic characterization of diverse rare and private spliceosomal mutations to infer their likely disease relevance. We utilized isogenic cell lines and primary patient materials to discover that 11 of 14 studied rare and private mutations in SRSF2 and U2AF1 induced distinct splicing alterations, including partially or completely phenocopying the alterations in exon and splice site recognition induced by hotspot mutations or driving "dual" phenocopies that mimicked two co-occurring hotspot mutations. Our data suggest that many rare and private spliceosomal mutations contribute to disease pathogenesis and illustrate the utility of molecular assays to inform precision medicine by inferring the potential disease relevance of newly discovered mutations.

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Aberrant RNA Splicing in Cancer

Annual Review of Cancer Biology ◽

10.1146/annurev-cancerbio-030617-050407 ◽

2019 ◽

Vol 3 (1) ◽

pp. 167-185 ◽

Cited By ~ 14

Author(s):

Luisa Escobar-Hoyos ◽

Katherine Knorr ◽

Omar Abdel-Wahab

Keyword(s):

Small Molecules ◽

Rna Splicing ◽

Clinical Development ◽

Splicing Factors ◽

Global Changes ◽

Splicing Regulation ◽

Enzymatic Process ◽

Patients With Cancer ◽

Aberrant Rna

RNA splicing, the enzymatic process of removing segments of premature RNA to produce mature RNA, is a key mediator of proteome diversity and regulator of gene expression. Increased systematic sequencing of the genome and transcriptome of cancers has identified a variety of means by which RNA splicing is altered in cancer relative to normal cells. These findings, in combination with the discovery of recurrent change-of-function mutations in splicing factors in a variety of cancers, suggest that alterations in splicing are drivers of tumorigenesis. Greater characterization of altered splicing in cancer parallels increasing efforts to pharmacologically perturb splicing and early-phase clinical development of small molecules that disrupt splicing in patients with cancer. Here we review recent studies of global changes in splicing in cancer, splicing regulation of mitogenic pathways critical in cancer transformation, and efforts to therapeutically target splicing in cancer.

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Splicing factor mutations in hematologic malignancies

Blood ◽

10.1182/blood.2019004260 ◽

2021 ◽

Author(s):

Sisi Chen ◽

Salima Benbarche ◽

Omar Abdel-Wahab

Keyword(s):

Rna Splicing ◽

Messenger Rna ◽

Hematologic Malignancies ◽

Splicing Factor ◽

Splicing Factors ◽

Loss Of Function ◽

Genes Encoding ◽

Precursor Rna ◽

Early Phase Clinical Trials ◽

Splicing Machinery

Mutations in genes encoding RNA splicing factors were discovered nearly ten years ago and are now understood to be amongst the most recurrent genetic abnormalities in patients with all forms of myeloid neoplasms and several types of lymphoproliferative disorders as well as subjects with clonal hematopoiesis. These discoveries implicate aberrant RNA splicing, the process by which precursor RNA is converted into mature messenger RNA, in the development of clonal hematopoietic conditions. Both the protein as well as the RNA components of the splicing machinery are affected by mutations at highly specific residues and a number of these mutations alter splicing in a manner distinct from loss of function. Importantly, cells bearing these mutations have now been shown to generate mRNA species with novel aberrant sequences, some of which may be critical to disease pathogenesis and/or novel targets for therapy. These findings have opened new avenues of research to understand biological pathways disrupted by altered splicing. In parallel, multiple studies have revealed that cells bearing change-of-function mutation in splicing factors are preferentially sensitized to any further genetic or chemical perturbations of the splicing machinery. These discoveries are now being pursued in several early phase clinical trials using molecules with diverse mechanisms of action. Here we review the molecular effects of splicing factor mutations on splicing, mechanisms by which these mutations drive clonal transformation of hematopoietic cells, and the development of new therapeutics targeting these genetic subsets of hematopoietic malignancies.

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U2AF1 mutations alter splice site recognition in hematological malignancies

10.1101/001107 ◽

2013 ◽

Author(s):

Janine O Ilagan ◽

Aravind Ramakrishnan ◽

Brian Hayes ◽

Michele E Murphy ◽

Ahmad S Zebari ◽

...

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Hematological Malignancies ◽

Normal Function ◽

Site Preference ◽

Sequencing Studies ◽

Genes Encoding ◽

Quantitative Changes ◽

Site Recognition

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.

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The Function of the Mutant p53-R175H in Cancer

Cancers ◽

10.3390/cancers13164088 ◽

2021 ◽

Vol 13 (16) ◽

pp. 4088

Author(s):

Yen-Ting Chiang ◽

Yi-Chung Chien ◽

Yu-Heng Lin ◽

Hui-Hsuan Wu ◽

Dung-Fang Lee ◽

...

Keyword(s):

Cancer Cells ◽

Gene Mutations ◽

Metabolic Reprogramming ◽

Cancer Development ◽

Mutant P53 ◽

Missense Mutations ◽

Wild Type ◽

Downstream Signaling ◽

Hotspot Mutations ◽

Wild Type P53

Wild-type p53 is known as “the guardian of the genome” because of its function of inducing DNA repair, cell-cycle arrest, and apoptosis, preventing the accumulation of gene mutations. TP53 is highly mutated in cancer cells and most TP53 hotspot mutations are missense mutations. Mutant p53 proteins, encoded by these hotspot mutations, lose canonical wild-type p53 functions and gain functions that promote cancer development, including promoting cancer cell proliferation, migration, invasion, initiation, metabolic reprogramming, angiogenesis, and conferring drug resistance to cancer cells. Among these hotspot mutations, p53-R175H has the highest occurrence. Although losing the transactivating function of the wild-type p53 and prone to aggregation, p53-R175H gains oncogenic functions by interacting with many proteins. In this review, we summarize the gain of functions of p53-R175H in different cancer types, the interacting proteins of p53-R175H, and the downstream signaling pathways affected by p53-R175H to depict a comprehensive role of p53-R175H in cancer development. We also summarize treatments that target p53-R175H, including reactivating p53-R175H with small molecules that can bind to p53-R175H and alter it into a wild-type-like structure, promoting the degradation of p53-R175H by targeting heat-shock proteins that maintain the stability of p53-R175H, and developing immunotherapies that target the p53-R175H–HLA complex presented by tumor cells.

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RUNX1/RUNX1T1 mediates alternative splicing and reorganises the transcriptional landscape in leukemia

Nature Communications ◽

10.1038/s41467-020-20848-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Vasily V. Grinev ◽

Farnaz Barneh ◽

Ilya M. Ilyushonak ◽

Sirintra Nakjang ◽

Job Smink ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Splicing ◽

Leukemic Cells ◽

Splicing Factors ◽

Domain Structures ◽

Indirect Control ◽

Expression Of Genes ◽

Alternative Transcription ◽

Genes Encoding ◽

Alternative Structure

AbstractThe fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of acute myeloid leukemia. Here we show that the RUNX1/RUNX1T1 oncogene is a regulator of alternative RNA splicing in leukemic cells. The comprehensive analysis of RUNX1/RUNX1T1-associated splicing events identifies two principal mechanisms that underlie the differential production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start site selection, and (ii) direct or indirect control of the expression of genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5’-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals alternative splicing as an important component of transcriptome re-organization in leukemia by an aberrant transcriptional regulator.

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Isolation and characterization of the genes encoding antimicrobial neutrophil cationic peptides, defensins.

Ensho ◽

10.2492/jsir1981.15.33 ◽

1995 ◽

Vol 15 (1) ◽

pp. 33-41

Author(s):

Isao Nagaoka ◽

Noriko Ishihara ◽

Akimasa Someya ◽

Kazuhisa Iwabuchi ◽

Shin Yomogida ◽

...

Keyword(s):

Cationic Peptides ◽

Isolation And Characterization ◽

Genes Encoding

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Protein complex formation in methionine chain-elongation and leucine biosynthesis

Scientific Reports ◽

10.1038/s41598-021-82790-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Li-Qun Chen ◽

Shweta Chhajed ◽

Tong Zhang ◽

Joseph M. Collins ◽

Qiuying Pang ◽

...

Keyword(s):

Protein Complexes ◽

Complete Characterization ◽

Bimolecular Fluorescence Complementation ◽

Chain Elongation ◽

Substrate Channeling ◽

Leucine Biosynthesis ◽

Protein Complex Formation ◽

Genes Encoding ◽

Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

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Fibrillin-1 gene mutations in a Chinese cohort with congenital ectopia lentis: spectrum and genotype–phenotype analysis

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2021-319084 ◽

2021 ◽

pp. bjophthalmol-2021-319084

Author(s):

Zexu Chen ◽

Tianhui Chen ◽

Min Zhang ◽

Jiahui Chen ◽

Michael Deng ◽

...

Keyword(s):

Calcium Binding ◽

Gene Mutations ◽

Mutation Spectrum ◽

Missense Mutations ◽

Ectopia Lentis ◽

Phenotype Analysis ◽

Chinese Cohort ◽

Fibrillin 1 ◽

Epidermal Growth ◽

Dependent Probe

AimsTo identify the mutation spectrum and genotype–phenotype correlations of fibrillin-1 (FBN1) mutations in a Chinese cohort with congenital ectopia lentis (EL).MethodsPatients clinically suspected of congenital zonulopathy were screened using panel-based next-generation sequencing followed by multiplex ligation-dependent probe amplification. All the probands were subjected to thorough ocular examinations. Molecular and clinical data were integrated in pursuit of genotype–phenotype correlation.ResultsA total of 131 probands of FBN1 mutations from unrelated families were recruited. Around 65% of the probands were children younger than 9 years old. Overall, 110 distinct FBN1 mutations were identified, including 39 novel ones. The most at-risk regions were exons 13, 2, 6, 15, 24 and 33 in descending order of mutation frequency. The most prevalent mutation was c.184C>T (seven, 5.34%) in the coding sequence and c.5788+5G>A (three, 2.29%) in introns. Missense mutations were the most frequent type (103, 78.63%); half of which were distributed in the N-terminal regions (53, 51.46%). The majority of missense mutations were detected in one of the calcium-binding epidermal growth factor-like domains (62, 60.19%), and 39 (62.90%) of them were substitutions of conserved cysteine residues. Microspherophakia (MSP) was found in 15 patients (11.45%). Mutations in the middle region (exons 22–42), especially exon 26, had higher risks of combined MSP (OR, 5.51 (95% CI 1.364 to 22.274), p=0.017).ConclusionsThis study extended the knowledge of the FBN1 mutation spectrum and provided novel insights into its clinical correlation regarding EL and MSP in the Chinese population.

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Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04185-7 ◽

2021 ◽

Author(s):

Fatma Ben Abid ◽

Clement K. M. Tsui ◽

Yohei Doi ◽

Anand Deshmukh ◽

Christi L. McElheny ◽

...

Keyword(s):

Common Species ◽

Clinical Samples ◽

Whole Genome ◽

E Coli ◽

Carbapenem Resistant ◽

Genes Encoding ◽

Encoding Genes ◽

Sequence Types ◽

Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

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Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in the Shenzhen Population

Human Heredity ◽

10.1159/000516808 ◽

2021 ◽

pp. 1-7

Author(s):

Jian Gao ◽

Sheng Lin ◽

Shiguo Chen ◽

Qunyan Wu ◽

Kaifeng Zheng ◽

...

Keyword(s):

G6pd Deficiency ◽

Amplification Refractory Mutation System ◽

Gene Variants ◽

Coding Region ◽

G6pd Gene ◽

Mutation System ◽

Hotspot Mutations ◽

Glucose 6 Phosphate Dehydrogenase ◽

Generation Sequencing

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. This study aimed to characterize the G6PD gene variant distribution in Shenzhen of Guangdong province. Methods: A total of 33,562 individuals were selected at the hospital for retrospective analysis, of which 1,213 cases with enzymatic activity-confirmed G6PD deficiency were screened for G6PD gene variants. Amplification refractory mutation system PCR was first used to screen the 6 dominant mutants in the Chinese population (c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, c.392G>T, and c.871G>A). If the 6 hotspot variants were not found, next-generation sequencing was then performed. Finally, Sanger sequencing was used to verify all the mutations. Results: The incidence of G6PD deficiency in this study was 3.54%. A total of 26 kinds of mutants were found in the coding region, except for c.-8-624T>C, which was in the noncoding region. c.1376G>T and c.1388G>A, both located in exon 12, were the top 2 mutants, accounting for 68.43% of all individuals. The 6 hotspot mutations had a cumulative proportion of 94.02%. Conclusions: This study provided detailed characteristics of G6PD gene variants in Shenzhen, and the results would be valuable to enrich the knowledge of G6PD deficiency.

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