scholarly journals RNA Splicing Defects in Cancer-Linked Genes Indicate Mutation or Focal Gene Deletion and Are Associated with TKI Resistance in CML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 662-662
Author(s):  
Naranie Shanmuganathan ◽  
Daniel Thomson ◽  
Carol Wadham ◽  
Verity A Saunders ◽  
Nur Hezrin Shahrin ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Research Funding ◽  
Cancer Genes ◽  
Aberrant Splicing ◽  
Cryptic Exon ◽  
Large Deletions ◽  
Tki Resistance ◽  
Splice Junctions ◽  
Runx1 Mutation

Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.

scholarly journals Mutations in the RNA Splicing Factor SF3B1 Promote Transformation through MYC Stabilization

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 882-882
Author(s):  
Akihide Yoshimi ◽  
Zhaoqi Liu ◽  
Wang Jiguang ◽  
Hana Cho ◽  
Stanley C Lee ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Research Funding ◽  
Double Mutant ◽  
Splicing Factor ◽  
Rna Seq ◽  
Aberrant Splicing ◽  
Fty 720 ◽  
The Impact ◽  
Mutant Cells

Abstract Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been identified as promoting use of aberrant 3' splice sites (3'ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas (Figure A, B). Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program. We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo. To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3'ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3'ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H). Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC. Figure. Figure. Disclosures Mato: Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.

High Incidence of Mutated Cancer-Associated Genes at Diagnosis in CML Patients with Early Transformation to Blast Crisis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 600-600 ◽  
Author(s):  
Susan Branford ◽  
Paul PS Wang ◽  
Wendy Tara Parker ◽  
David Yeung ◽  
Justine E Marum ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Epigenetic Regulation ◽  
Splice Site ◽  
Nuclear Export ◽  
Research Funding ◽  
Blast Crisis ◽  
Chromatin Modification ◽  
Cancer Genes ◽  
First Line ◽  
Advisory Committees

Abstract Background: A single genomic event is sufficient to cause CML; Ph translocation and the resulting BCR-ABL fusion. Additional genomic lesions accompany progression, which occurs very rapidly after diagnosis (dx) in a minority. Identification at dx of patients (pts) with poor prognosis remains an important goal and new sequencing technology enhances the prospects of uncovering pathologically relevant lesions for early warning of disease progression. Aim: To determine the somatic genomic landscape at dx, the risk conferred by genomic lesions towards blast crisis (BC), and whether mechanisms that underlie CML progression are shared by other malignancies. Method: Sequencing the whole exome (WES) and transcriptome (RNAseq) of paired tumor-normal samples (bone marrow mesenchymal stromal cells or remission) identified somatic single nucleotide variants, indels and gene fusions. Twenty-eight chronic phase (CP) first line imatinib (IM) treated pts were tested: 14 had BC at a median of 9 mo, r 3-60; and 14 had good response (MMR by 6 mo). Also tested were 4 pts diagnosed in advanced phase (2 accelerated phase [AP]; 2 BC) and 5 historical pts with BC at a median of 64 mo, r 38-100. Results: At dx, a median of 33 somatic variants were detected per pt (r 1-62). The number of variants did not correlate with response, or CP vs AP/BC, but increased with age (r =0.48, P =.007), consistent with accumulation of variants in stem cells with aging and suggesting that many may be "passenger mutations". Non synonymous protein coding variants were present at a median of 7 per pt at dx (r 0-17), again without difference between groups. Most variants had an allele frequency close to 50%, indicating their likely presence in all leukemic cells. However, polyclonality at dx was evident by variants with low allele frequency that either expanded or diminished at BC, Fig A. All 4 pts in AP/BC at dx had mutations in genes implicated in cancer pathogenesis (cancer genes) at dx; CBFB-MYH11 fusion, BCORL1, GATA2 and PTPRT, and SMARCA1. Of the 28 pts with first line IM, 11 had 15 somatic and 1 germline non synonymous variants/fusions at dx of known/potential significance: oncogenic mutations in IDH1 (R132H) and TP53 (germline R248Q); 6 frameshift/stop/splice site mutations in ASXL1, 1 EZH2 stop, 1 SETD1B stop, 1 MLL2 frameshift, 1 CHD1 splice site; and 4 novel fusions. Two of the fusions were generated by inversions of 2-13 MB of chr 22: PPM1F-SPECC1L (truncating the protein phosphatase PPM1F) and MYH9-BCR (truncating MYH9, reported to regulate p53 stability). Of these 11 pts, 9 had BC at a median of 6 mo of IM, r 3-39, and 2 had MMR by 6 mo. The 2 good response pts had ASXL1 mutations (both stop) and 1 also had a fusion involving chr 9 and 22 (TNRC6B-NEK6). The frequency of BC in CP pts with potentially pathogenic variants at dx was significantly higher than pts without such variants; 9/11 (82%) vs 5/17 (29%), P =.02. At BC, 18 pts had WES performed. A median of 6 non synonymous variants were gained (r 0-15) including 1-4 mutations in cancer genes in 15/18 pts, Fig B. Six of 13 first line IM pts also had 11 BCR-ABL KD mutations at BC (8 P loop) and 5/6 were among the pts who acquired mutations in cancer genes. The pt with the germline oncogenic TP53 mutation acquired a novel ANKRD11-UBQLN1 fusion at BC at 5 mo. Interestingly, ANKRD11 is a key regulator of the oncogenic potential of this mutation. In total, 6 genes were recurrently mutated; ASXL1, BCORL1, RUNX1, GATA2, MLL and UBE2A. Mutations occurred in genes that primarily belonged to classes mutated in AML, Fig B. Of the 23 AP/BC samples, 22 had non synonymous variants in genes involved in epigenetic regulation/chromatin modification. Variants were also detected in genes involved in ubiquitination and nuclear export, including an XPO1 variant reported in CLL. Nucleocytoplasmic transport has been implicated in IM resistance. Conclusion: Risk of BC was significantly associated with cancer gene mutation or novel fusions at dx. Some mutated pathways in CML were common to other cancers. Epigenetic regulation/chromatin modification appears to play a central role in CML pathogenesis, and ubiquitination and nuclear export may be of emerging relevance. Notably, most pts with BCR-ABL KD mutations at BC also acquired cancer associated mutations, indicating multiple mechanisms may contribute to progression. Future testing at dx will likely include tumor/normal exome/transcriptome sequencing to aid risk stratification. Figure 1. Figure 1. Disclosures Branford: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. Yeung:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals An RNA-Based Next Generation Sequencing (NGS) Strategy Detects More Cancer Gene Mutations Than a DNA-Based Approach for the Prediction and Assessment of Resistance in CML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2918-2918
Author(s):  
Susan Branford ◽  
Carol Wadham ◽  
Naranie Shanmuganathan ◽  
Daniel Thomson ◽  
NUR Hezrin Shahrin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Allele Frequency ◽  
Cell Lines ◽  
Rna Splicing ◽  
Research Funding ◽  
Gene Fusions ◽  
Cancer Gene ◽  
Functional Effect ◽  
Gene Deletions ◽  
Splice Junctions

Background Mutation of genes linked to hematologic cancer have recently been reported in CML and are associated with early progression and resistance (Reviewed in Branford, Kim Leuk 2019). The mutations comprise single nucleotide variants (SNVs) and small insertions/deletions (indels), plus gene fusions and large focal gene deletions. In 39 patients (pts) in blast crisis (BC), all had at least 1 cancer gene mutation, including fusions in 33%: partner genes MLL, RUNX1, IKZF1, MECOM and CBFB. 50% of the fusions were novel and some were present at chronic phase diagnosis. BCR-ABL1 mutations rarely occurred as the sole mutant. NGS offers critical information for resistance assessment. For many clinical purposes, targeted DNA sequencing (seq) using panels of specific disease related genes is the most cost effective screening choice. However, this strategy could miss relevant fusions and deletions. Aim To determine whether an RNA based approach is more informative than DNA for detecting a broad range of mutations. Method A hybridization capture NGS gene panel was developed to target 126 genes relevant for myeloid/lymphoid leukemia. In a pilot study, DNA and RNA derived from 5 leukemia cell lines with well characterized mutations, including fusions and deletions, were panel sequenced. An additional 6 cell lines were sequenced using RNA, plus 49 pt samples with RNA stored for up to 14.6 years: 45 at diagnosis and 4 at BC/resistance. Six of these pt samples had prior whole exome and/or whole transcriptome seq. We used total RNA that detected intronic splice region variants from pre-spliced RNA. SNVs/indels were called from DNA/RNA with FreeBayes. Manta called focal deletions from DNA. Known and novel RNA fusions and novel splice junctions were detected using the STAR aligner. Gene expression used edgeR. Results For the 5 cell lines with DNA versus (v) RNA seq, SNVs/indels were reliably called in RNA, with a strong positive correlation of mutant allele frequency: DNA v RNA, r = 0.93. Two TP53 small deletions of 26 and 46 bp were not called in RNA, but were instead detected as novel RNA splice junctions. Read counts were 5.2 fold higher for RNA than DNA at sites of clinically relevant mutants, consistent with enrichment of seq read depth proportional to expression. Overall, RNA revealed a higher number of relevant mutants than DNA: RNA = 49 v DNA = 37, Fig A-C. Notably, the functional effect of splice region disrupting mutants and large focal deletions were evident by novel RNA splicing, Fig D-F. In the total 11 cell lines tested with RNA, all 13 reported fusions were called, including BCR-ABL1 and RUNX1, MLL, ETV6 and CBFB fusions. For 7 cell lines with variants described in the COSMIC Cell Lines Project, 23/23 cancer gene SNVs/indels were called, plus 7 cancer gene SNVs/indels not reported. These were verified by DNA seq. 15 gene deletions were evident by atypical RNA splicing and verified by DNA seq: IKZF1, CDKN2A/B, PAX5, BTG1, RB1 and NCOR1. Five other cell lines had verified CDKN2A deletions that were evident by loss of gene expression, Fig G. Two BTG1 deletions were not detected. For the 6 pt samples re-sequenced by the RNA panel, 8/8 verified fusion transcripts were detected with a 31 fold enrichment of read counts. 11/11 cancer gene SNVs/indels were called and 3/4 gene deletions. The exception was a CDKN2A deletion not detected by novel splicing but evident as loss of expression, Fig G. Seven other cancer gene SNVs were found at low allele frequency, including a resistant BCR-ABL1 mutation at 1.7% in the oldest sample. Of the 43 diagnosis samples without prior NGS, BCR-ABL1 transcripts were detected in all. BCR-ABL1 genomic breakpoints were called at base pair resolution in 39, 91%. Two pts had mutated ASXL1 at diagnosis and both failed imatinib by 9 months with mutant BCR-ABL1. By gene expression analysis, all but 1 of the total 45 diagnosis samples clustered together. The exception was a pt who transformed to lymphoid BC at 6 months that clustered with the lymphoid cell lines and lymphoid BC pts, Fig H. Conclusion RNA gene panel seq demonstrated enhanced sensitivity and an increased yield of clinically relevant mutations compared with DNA panel seq. A single RNA assay has the capacity to detect SNV/indels, known and novel gene fusions, focal deletions and the likely functional effect of splice disrupting mutations. RNA panel seq is a valuable tool for the comprehensive assessment of mutations that drive CML treatment failure and drug resistance. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Shanmuganathan:Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel; Novartis, Bristol-Myers Squibb, Celgene: Research Funding.

scholarly journals ZRSR2 Mutations Cause Dysregulated RNA Splicing in MDS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4609-4609
Author(s):  
Vikas Madan ◽  
Deepika Kanojia ◽  
Li Jia ◽  
Ryoko Okamoto ◽  
Aiko Sato-Otsubo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Splice Site ◽  
Rna Splicing ◽  
Somatic Mutations ◽  
Rna Binding ◽  
Ectopic Expression ◽  
Leukemia Cell ◽  
Aberrant Splicing ◽  
Differentiation Potential ◽  
Inactivating Mutations

Abstract Recurrent somatic mutations have been uncovered in several components of the spliceosome in Myelodysplastic Syndrome (MDS). Recent high throughput sequencing of large cohorts of MDS has established RNA splicing as the pathway most frequently targeted by somatic mutations. These findings implicate dysregulated RNA splicing in the pathogenesis of MDS. However, the mechanism linking aberrant splicing to the development of MDS is unknown. ZRSR2, a frequently mutated spliceosome gene in MDS is located on the X chromosome. Somatic alterations of ZRSR2 are typically inactivating mutations (frameshift indels, nonsense point mutations or splice site mutations) which are observed predominantly in males. Mutations in ZRSR2are more prevalent in MDS subtypes without ring sideroblasts and chronic myelomonocytic leukemia (CMML) and are associated with elevated bone marrow blasts and higher rate of progression to AML. Although ZRSR2 has been suggested to interact with other splice proteins, U2AF2 and SRSF2, at the 3΄ splice sites during the pre-spliceosome assembly, its precise role in RNA splicing remains unexplored. In this study, we demonstrate that deficiency of ZRSR2 leads to impaired splicing of U12-type introns which are dependent upon the minor spliceosome. RNA-Sequencing of MDS bone marrow harboring inactivating mutations of ZRSR2 revealed aberrant retention of U12-type introns as compared to bone marrow with wild-type ZRSR2. In addition, several U12-type introns displayed mis-splicing associated with recognition of cryptic splice-site. In contrast, the splicing of U2-type introns (dependent upon the major spliceosome) was largely unaffected. The mis-spliced introns were found in several key genes including PTEN, MAPK1, MAPK3, BRAF and E2F2, and the impaired splicing of several introns in ZRSR2 mutant bone marrow was validated experimentally. Further, short hairpin RNA (shRNA) mediated knockdown of ZRSR2 in MDS/AML TF1 cells led to impaired splicing specifically of U12-type introns. We also observe that ectopic expression of ZRSR2 in stable knockdown 293T cells resulted in increase in splicing efficiency of U12-type introns. Further, the downregulation of ZRSR2 in leukemia cells results in reduced growth and clonogenic potential in leukemia cell lines. Moreover, knockdown of ZRSR2 in human CD34+ cells displayed altered differentiation potential towards erythroid and myeloid lineages in vitro. Overall, the dysregulated RNA splicing of U12-type introns in ZRSR2 mutant samples affects several crucial genes involved in cell cycle, signaling, RNA binding and transport. These genes are potential mediators of MDS phenotype. Ours is a first study which demonstrates the functional consequences of ZRSR2 mutations in MDS and identifies a specific role of ZRSR2 in regulating RNA splicing. It underlines aberrant splicing of U12-type introns as a distinctive feature of ZRSR2 mutant MDS. Disclosures Kohlmann: MLL Munich Leukemia Laboratory, Munich, Germany: Employment. Grossmann:MLL Munich Leukemia Laboratory, Munich, Germany: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Insights of Non-canonical Splice-site Variants on RNA Splicing in Patients with Congenital Hypothyroidism

2021 ◽  
Author(s):  
Najla Albader ◽  
Minjing Zou ◽  
Huda A BinEssa ◽  
Saba Abdi ◽  
Anwar F Al-Enezi ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Next Generation Sequencing Data ◽  
Splice Sites ◽  
Sequencing Data ◽  
Aberrant Splicing ◽  
Hormone Synthesis ◽  
Generation Sequencing

Abstract Context Congenital hypothyroidism (CH) is caused by mutations in the genes for thyroid hormone synthesis. In our previous investigation of CH patients, ~53% of patients had mutations in either coding exons or canonical splice-sites of causative genes. Non-canonical splice-sites variants in the intron were detected but their pathogenic significance was not known. Objective To evaluate non-canonical splice-site variants on pre-mRNA splicing of CH-causing genes. Methods Next-generation sequencing data of 55 CH cases in 47 families were analyzed to identify rare intron variants. The effects of variants on pre-mRNA splicing were investigated by minigene RNA-splicing assays. Results Four intron variants were found in 3 patients: SLC26A4 c.1544 + 9C>T and c.1707 + 94C>T in one patient, and SLC5A5 c.970-48G>C and c.1652-97A>C in two other patients. The c.1707 + 94C>T and c.970-48G>C caused exons 15 and 16 skipping, and exon 8 skipping, respectively. The remaining variants had no effect on RNA splicing. Furthermore, we analyzed 28 previously reported non-canonical splice-site variants (4 in TG and 24 in SLC26A4). Among them, 15 variants (~54%) resulted in aberrant splicing and 13 variants had no effect on RNA splicing. These data were compared with three variant-prediction programs (FATHMM-XF, FATHMM-MKL, and CADD). Among 32 variants, FATHMM-XF, FATHMM-MKL, and CADD correctly predicted 20 (63%), 17 (53%), and 26 (81%) variants, respectively. Conclusions Two novel deep intron mutations have been identified in SLC26A4 and SLC5A5, bringing the total number of solved families with disease-causing mutations to ~45% in our cohort. Approximately 46% (13/28) reported non-canonical splice-site mutations do not disrupt pre-mRNA splicing. CADD provides highest prediction accuracy of non-canonical splice-site variants.

scholarly journals Elucidation of the aberrant 3′ splice site selection by cancer-associated mutations on the U2AF1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hisashi Yoshida ◽  
Sam-Yong Park ◽  
Gyosuke Sakashita ◽  
Yuko Nariai ◽  
Kanako Kuwasako ◽  
...  
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Rna Splicing ◽  
Single Amino Acid ◽  
Splice Site Selection ◽  
Aberrant Splicing ◽  
Serious Disease ◽  
New Treatments ◽  
Target Rna ◽  
Single Amino Acid Mutation

Abstract The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

scholarly journals Regulation of RNA Splicing: Aberrant Splicing Regulation and Therapeutic Targets in Cancer

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 923
Author(s):  
Koji Kitamura ◽  
Keisuke Nimura
Keyword(s):  
Dna Sequences ◽  
Rna Splicing ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Aberrant Splicing ◽  
Aberrant Expression ◽  
Non Coding Rnas ◽  
Precursor Mrna ◽  
Splicing Enhancer ◽  
Small Nuclear Ribonucleoproteins

RNA splicing is a critical step in the maturation of precursor mRNA (pre-mRNA) by removing introns and exons. The combination of inclusion and exclusion of introns and exons in pre-mRNA can generate vast diversity in mature mRNA from a limited number of genes. Cancer cells acquire cancer-specific mechanisms through aberrant splicing regulation to acquire resistance to treatment and to promote malignancy. Splicing regulation involves many factors, such as proteins, non-coding RNAs, and DNA sequences at many steps. Thus, the dysregulation of splicing is caused by many factors, including mutations in RNA splicing factors, aberrant expression levels of RNA splicing factors, small nuclear ribonucleoproteins biogenesis, mutations in snRNA, or genomic sequences that are involved in the regulation of splicing, such as 5’ and 3’ splice sites, branch point site, splicing enhancer/silencer, and changes in the chromatin status that affect the splicing profile. This review focuses on the dysregulation of RNA splicing related to cancer and the associated therapeutic methods.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

1988 ◽  
Vol 8 (4) ◽  
pp. 1558-1569
Author(s):  
P E Cizdziel ◽  
M de Mars ◽  
E C Murphy
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Exon Ligation ◽  
Branch Point Sequence ◽  
Infected Cells ◽  
Low Efficiency

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

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