scholarly journals Expression changes confirm genomic variants predicted to result in allele-specific, alternative mRNA splicing

2019 ◽  
Author(s):  
Eliseos J. Mucaki ◽  
Ben C. Shirley ◽  
Peter K. Rogan
Keyword(s):  
Gene Expression ◽  
Information Theory ◽  
Splice Site ◽  
Mrna Splicing ◽  
Splice Sites ◽  
Rt Pcr ◽  
Alternative Mrna Splicing ◽  
Site Use ◽  
Allele Specific ◽  
Specific Alternative

AbstractSplice isoform structure and abundance can be affected by either non-coding or masquerading coding variants that alter the structure or abundance of transcripts. When these variants are common in the population, these non-constitutive transcripts are sufficiently frequent so as to resemble naturally occurring, alternative mRNA splicing. Prediction of the effects of such variants has been shown to be accurate using information theory-based methods. Single nucleotide polymorphisms (SNPs) predicted to significantly alter natural and/or cryptic splice site strength were shown to affect gene expression. Splicing changes for known SNP genotypes were confirmed in HapMap lymphoblastoid cell lines with gene expression microarrays and custom designed q-RT-PCR or TaqMan assays. The majority of these SNPs (15 of 22) as well as an independent set of 24 variants were then subjected to RNAseq analysis using the ValidSpliceMut web beacon (http://validsplicemut.cytognomix.com), which is based on data from the Cancer Genome Atlas and International Cancer Genome Consortium. SNPs from different genes analyzed with gene expression microarray and q-RT-PCR exhibited significant changes in affected splice site use. Thirteen SNPs directly affected exon inclusion and 10 altered cryptic site use. Homozygous SNP genotypes resulting in stronger splice sites exhibited higher levels of processed mRNA than alleles associated with weaker sites. Four SNPs exhibited variable expression among individuals with the same genotypes, masking statistically significant expression differences between alleles. Genome-wide information theory and expression analyses (RNAseq) in tumour exomes and genomes confirmed splicing effects for 7 of the HapMap SNP and 14 SNPs identified from tumour genomes. q-RT-PCR resolved rare splice isoforms with read abundance too low for statistical significance in ValidSpliceMut. Nevertheless, the web-beacon provides evidence of unanticipated splicing outcomes, for example, intron retention due to compromised recognition of constitutive splice sites. Thus, ValidSpliceMut and q-RT-PCR represent complementary resources for identification of allele-specific, alternative splicing.

Allele-Specific Effects Of U2AF1 Mutations On Alternative Splicing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2748-2748
Author(s):  
Theresa Okeyo-Owuor ◽  
Brian S. White ◽  
Dipika Mohan ◽  
Malachi Griffith ◽  
Matthew J. Walter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Mrna Splicing ◽  
Rt Pcr ◽  
Specific Effects ◽  
Cd34 Cells ◽  
Recurrent Mutations ◽  
Allele Specific ◽  
Splice Junctions ◽  
Alternative Isoforms

Abstract Four independent groups, including ours, recently discovered recurrent mutations in core components of the pre-mRNA splicing complex (the “spliceosome”) in myelodysplastic syndrome (MDS) patient samples using next-generation sequencing approaches. We previously identified mutations in U2AF1 affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of patients with de novo MDS. Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS. To determine the effects of S34 and Q157 mutations on U2AF1 splicing activity, we utilized GH1 and FMR1 minigene assays, which we have previously used to assess alternative splicing. Here, we report that recurrent mutations in U2AF1 have allele-specific effects on alternative splicing for both GH1 and FMR1 minigenes when transiently transfected in 293T cells. The U2AF1 S34F mutant allele yielded the most significant increase in alternative splicing activity for both GH1 and FMR1 (GH1: p<0.01; FMR1: p<0.001; n>3 biological replicates), compared to wildtype (WT). The S34Y allele modestly enhanced expression of the alternative isoforms for both minigenes (GH1: p<0.05; FMR1: p<0.01). Conversely, we saw a reduction in expression of the alternative isoforms in cells expressing the Q157R and Q157P alleles compared with WT (p<0.01) or the S34 alleles (p<0.001). In cells expressing the S34F/Q157R mutant (in which both the S34F and Q157R mutations occur on one allele, discovered in one patient with MDS), GH1 and FMR1 splicing was indistinguishable from WT. We then assessed the effect of the S34F mutation on U2AF1 sub-cellular localization and protein interaction within the spliceosome. Using fluorescence immunocytochemistry in transfected 293T cells, we found that S34F U2AF1 localized normally within the nuclear speckles and co-localized with U2AF2 and SRSF2. However, immunoprecipitation of epitope-tagged constructs transfected in K562 cells demonstrated that S34F U2AF1 had reduced direct interaction with U2AF2, compared to WT U2AF1. We next performed RNA-seq to comprehensively determine the effects of the S34F mutation on pre-mRNA splicing and gene expression. We transfected primary human CD34+ cells (derived from cord blood) with constructs expressing either WT or S34F U2AF1 (3 biological replicates for each condition) and sorted for transfected (GFP positive) cells after 24 hours. cDNA libraries depleted of ribosomal RNA were sequenced on the Illumina platform. Transient overexpression of WT vs. S34F U2AF1 did not significantly alter global gene expression profiles (by Cufflnks FPKM) in an unsupervised analysis. Analyses of splice junctions using ALEXA-Seq and EdgeR revealed significant differences in the abundance of known splice junctions (S34F>WT: 104, S34F<WT: 188; FDR<0.05, fold change >4). We also detected expression differences at novel splice junctions resulting from use of either known or novel alternative splice acceptor and/or donor sites (S34F>WT: 135, S34F<WT: 229). Both the known and novel junctions reflect alterations in canonical splicing, exon skipping or retention, intron retention and cryptic site usage. We selected junctions with higher expression in S34F samples for validation in CD34+ cells using RT-PCR and gel electrophoresis. 16/27 genes with known junctions and 10/10 with novel junctions validated (p<0.05; n=3 biological replicates). 9 of the validated changes were tested in primary clinical samples by RT-PCR (n=6 MDS bone marrow with S34F U2AF1 vs. n=6 MDS controls with no U2AF1 splicing mutations) and 5 were confirmed (p<0.01), including alternative splicing involving known (DEK, SERPIN8B, KIAA1033, IFI44) and novel (ABI1) junctions. We conclude that the S34F mutation affects U2AF1 function, leading to aberrant alternative splicing of target genes. Whether alternative splicing perturbs hematopoiesis and contributes to MDS pathogenesis is not yet known. Disclosures: No relevant conflicts of interest to declare.

scholarly journals U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

1993 ◽  
Vol 13 (5) ◽  
pp. 2666-2676 ◽  
Author(s):  
J B Cohen ◽  
S D Broz ◽  
A D Levinson
Keyword(s):  
Splice Site ◽  
Mammalian Cells ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
Gene Complementation ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

scholarly journals hnRNP A1 Regulates Alternative Splicing of Tau Exon 10 by Targeting 3′ Splice Sites

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 936 ◽  
Author(s):  
Yongchao Liu ◽  
Donggun Kim ◽  
Namjeong Choi ◽  
Jagyeong Oh ◽  
Jiyeon Ha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Alternative Splicing ◽  
Splice Site ◽  
Hnrnp A1 ◽  
Gene Ontology Analysis ◽  
Splice Sites ◽  
Neuronal Function ◽  
Brain Functions ◽  
Exon 10

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3′ splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.

scholarly journals Regulation of Sex-Specific Selection offruitless 5′ Splice Sites by transformerand transformer-2

1998 ◽  
Vol 18 (1) ◽  
pp. 450-458 ◽  
Author(s):  
Volker Heinrichs ◽  
Lisa C. Ryner ◽  
Bruce S. Baker
Keyword(s):  
Splice Site ◽  
Courtship Behavior ◽  
Specific Pattern ◽  
Splice Sites ◽  
Repeat Elements ◽  
Specific Alternative ◽  
Female Specific ◽  
Male Specific ◽  
Splicing Enhancer

ABSTRACT In Drosophila melanogaster, the fruitless(fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specificfru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genestransformer (tra) and transformer-2(tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requirescis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. Thisfru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.

Characterization of a Novel Aberrant Splice Site, 79bp Downstream of Exon 5 in the Human Factor 7 Gene Detected in Patients with Severe Congenital Factor VII Deficiency.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3481-3481
Author(s):  
Karin Wulff ◽  
Jan Astermark ◽  
Falko F H Herrmann ◽  
Günther Auerswald ◽  
Winnie Schröder
Keyword(s):  
Splice Site ◽  
Donor Splice Site ◽  
Aberrant Splice ◽  
The Novel ◽  
Splice Sites ◽  
Wild Type ◽  
Rt Pcr ◽  
Donor Splice ◽  
Naturally Occurring ◽  
Fvii Deficiency

Abstract Abstract 3481 Poster Board III-418 Hereditary FVII deficiency (FVIID) is a rare congenital bleeding disorder with an estimated prevalence of symptomatic individuals of 1:500,000. In the “Greifswald Registry FVII Deficiency” molecular defects of more than 1000 FVII deficient patients were described. By direct sequencing of the F7 genes in congenital FVIID revealed 146 different F7 gene mutations including 25 different mutations (18% of all) in the naturally-occurring acceptor or donor splice sites (Tab.1) were identified. In seven FVIID patients from Sweden and Germany the novel lesion g.IVS5+78G>A - downstream of the naturally-occurring donor splice sites of exon 5 - was identified. This variation was detected heterozygous in FVIID patients with FVII: C levels of 15%, 27%, 31%, 40% and 65%, and FVII: Ag levels between 25-50%. In two compound heterozygous patients with FVII: C levels of 1% und FVII: Ag levels of 2% and 3% respectively, one well-known causative FVII mutation is combined with the novel lesion g.IVS5+78G>A. The influence of this novel F7 gene variation on splicing was investigated by RT-PCR analysis and in vitro expression studies using exon-trap vector constructs. The total RNA was isolated from peripheral leucocytes and analyzed by one step RT-PCR and sequencing. Fragments of exon 5 and a part of the flanking intron 5 region (g.7679 –g.8073) were amplified of patients' DNA and cloned into the exon trap-vector pET01. Different vector constructs containing minigenes of the wild type (g.IVS5+78G) or mutant form (g.IVS5+78A) and the corresponding minigenes with an “optimized” naturally-occurring donor splice site in position +5 respectively were transfected into HEK293 cells. The expressed RNA was isolated and characterized. Consensus Values (CV) for all donor splice sites were calculated using a splice site detection tool according Shapiro and Senapathy (1987). The RT-PCR analysis in patients indicate that the novel variation g.IVS5+78G>A in intron 5 created an aberrant splice site in position 79bp downstream of exon 5 even though the naturally-occurring donor-splice-site of exon 5 is not abolished. An insertion of 79bp of intron 5 into the mRNA leads to a frame shift and predicts a premature termination 11 codons past the last unaltered codon. Minigenes include the naturally-occurring splice site and the variation g.IVS5+78A used exclusively the aberrant splice position 79bp downstream of exon 5 whereas wild type minigenes with the naturally-occurring splice site and the wild type form g.IVS5+78G produced normally spliced mRNA. In a following experiment the “naturally-occurring splice site” of exon 5 was optimized by the additional substitution g.IVS5+5C>G which increased their CV from 76.6 to 90.9 compared to the CV of the novel mutant g.IVS5+78A of 80.3. In presence of both mutations (g.IVS5+5G and g.IVS5+78A) only normal spliced mRNA was expressed of this minigene. In this construct the mutation g.IVS5+78G>A was without importance for the mRNA splicing. The results of the in vitro experiments demonstrated, that the Consensus Values (CV) seems to be an important factor for the selection of donor splice sites in the F7 gene. In the “Greifswald Registry FVII Deficiency” 26 different splice site variations in F7 gene were identified (Tab. 1). The atypical splice site variation g.IVS5+78G>A, +78bp downstream of exon 5 was present in 7 FVIID patients from Sweden and Germany in different genotypes. This novel F7 gene mutation g.IVS5+78G>A creates an aberrant splice site in position +79 of intron 5 and predicts premature termination. RNA analysis and expression studies demonstrated, that this novel F7 gene lesion is a type I mutation with low FVII:C and FVII: Ag levels and is the basis defect in 7 FVIID patients of the “Greifswald Registry FVII Deficiency”. Tab. 1 26 different intronic F7 gene mutations analysed in FVII deficiency patients of the “Greifswald Registry FVII Deficiency” Intron Acceptor splice site Intron Donor splice site 1b g.IVS1b-11G>A 1a g.IVS1a+5G>A 1b *g.IVS1b8del14bp 1a *g.IVS1a+6T>G 1b *g.IVS1b-3C>G 1a *g.IVS1a+8C>T 2 *g.IVS2-3C>G 2 g.IVS2+1G>A 3 g.IVS3-1G>A 2 *g.IVS2+1G>T 3 *g-IVS3-1G>T 2 *g.IVS2+1G>C 4 *g.IVS4-7T>G 2 *g.IVS2+1delG 7 *g.IVS7-10T>C 2 g.IVS2+5G>T 7 *g.IVS7-3C>G 3 *g.IVS3+1G>T 7 *g.IVS7-1G>A 4 g.IVS4+1G>A 5 *g.IVS5+78G>A 6 *g.IVS6+1G>A 6 g.IVS6+1G>T 6 *g.IVS6+3A>G 7 *g.IVS7+1G>A 7 g.IVS7+3_6 del4bp * novel mutations (HGMD Factor 7 Database, 2009 /http://www.hgmd.org) Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 282 ◽  
Author(s):  
Natasha G. Caminsky ◽  
Eliseos J. Mucaki ◽  
Peter K. Rogan
Keyword(s):  
Splice Site ◽  
Genetic Disease ◽  
Mrna Splicing ◽  
Regulatory Sequences ◽  
Splice Sites ◽  
Review Of The Literature ◽  
Cryptic Splice Site ◽  
Broad Perspective ◽  
Genomic Variants ◽  
Splicing Mutations

The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations.

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