Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons

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.

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Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants

10.21203/rs.2.12748/v2 ◽

2019 ◽

Author(s):

Raphael Leman ◽

Hélène Tubeuf ◽

Sabine Raad ◽

Isabelle Tournier ◽

Céline Derambure ◽

...

Keyword(s):

Branch Point ◽

Mrna Splicing ◽

Large Set ◽

Branch Points ◽

Splice Sites ◽

Prediction Tools ◽

Mature Mrna ◽

Bioinformatics Tools ◽

The Impact

Abstract Background: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3’ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3’ss. Results: We used a large set of constitutive and alternative human 3’ss collected from Ensembl (n = 264,787 3’ss) and from in-house RNAseq experiments (n = 51,986 3’ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3’ss (99.48 % and 65.84 % accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17 %. Conclusions: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3’ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area. Keywords: Branch Point, Prediction, RNA, Benchmark, HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR, RNABPS, Variants

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Mechanisms for selecting 5′ splice sites in mammalian pre-mRNA splicing

Trends in Genetics ◽

10.1016/0168-9525(94)90233-x ◽

1994 ◽

Vol 10 (3) ◽

pp. 100-106 ◽

Cited By ~ 129

Author(s):

David S. Horowitz ◽

Adrian R. Krainer

Keyword(s):

Mrna Splicing ◽

Splice Sites

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Elimination of mRNA splicing by a point mutation outside the conserved GU at 5′ splice sites

Nucleic Acids Research ◽

10.1093/nar/12.9.3821 ◽

1984 ◽

Vol 12 (9) ◽

pp. 3821-3827 ◽

Cited By ~ 9

Author(s):

Craig Montell ◽

Arnold J. Berk

Keyword(s):

Point Mutation ◽

Mrna Splicing ◽

Splice Sites

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

F1000Research ◽

10.12688/f1000research.5654.1 ◽

2014 ◽

Vol 3 ◽

pp. 282 ◽

Cited By ~ 47

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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Potential G-quadruplex forming sequences and N6-methyladenosine colocalize at human pre-mRNA intron splice sites

10.1101/2020.02.07.939116 ◽

2020 ◽

Author(s):

Manuel Jara-Espejo ◽

Aaron M. Fleming ◽

Cynthia J. Burrows

Keyword(s):

Bioinformatic Analysis ◽

Mrna Splicing ◽

Published Data ◽

Splicing Factors ◽

Splice Sites ◽

Sequence Context ◽

Intron Splice ◽

Alternative Mrna Splicing ◽

G Quadruplex ◽

Splice Junctions

ABSTRACTUsing bioinformatic analysis of published data, we identify in human mRNA that potential G-quadruplex forming sequences (PQSs) colocalize with the epitranscriptomic modifications N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). A deeper analysis of the colocalized m6A and PQSs found them intronic in pre-mRNA near 5′ and 3′ splice sites. The loop lengths and sequence context of the m6A-bearing PQSs found short loops most commonly comprised of A nucleotides. This observation is consistent with literature reports of intronic m6A found in SAG (S = C or G) consensus motifs that are also recognized by splicing factors. The localization of m6A and PQSs in pre-mRNA at intron splice junctions suggests that these features could be involved in alternative mRNA splicing. A similar analysis for PQSs around sites of Ψ installation or A-to-I editing in mRNA also found a colocalization; however, the frequency was less than that observed with m6A.TOC Graphic

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Splicing Control in Normal and Aberrant Erythropoiesis

Blood ◽

10.1182/blood-2018-99-109414 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-47-SCI-47

Author(s):

Esther Obeng

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Iron Transport ◽

Rna Binding ◽

Intron Retention ◽

Point Mutations ◽

Cytoskeletal Proteins ◽

Mrna Splicing ◽

Splice Sites ◽

Erythroid Maturation

Abstract Alternative splicing is employed by all eukaryotic cells to increase proteome diversity and to regulate gene expression. RNA sequencing analysis of purified populations of erythroblasts at different stages of maturation has led to the identification of a dynamic alternative splicing program that directly modulates the protein isoform expression of cytoskeletal proteins and genes involved in RNA processing, heme biosynthesis, and iron transport. Regulated interactions of multiple RNA-binding proteins and cis-regulatory sequences located within exons or their flanking introns promote or inhibit functional spliceosome assembly at splice junctions, leading to altered exon inclusion or intron retention. Exon skipping regulates tissue and stage specific isoform expression of red cell membrane cytoskeletal proteins including EPB41, ankyrin, and band 3. Intron retention can lead to a frame shift during translation and introduction of a premature termination codon (PTC), that marks the transcript for degradation via the nonsense mediated decay pathway (NMD) upon export from the nucleus into the cytoplasm. Intron retention leading to posttranscriptional regulation of gene expression during terminal erythroid maturation has been identified in genes involved in RNA processing and iron transport including SF3B1, SNRNP70, SLC25A37 and SLC25A28. Mutations that alter mRNA splice sites or introduce PTCs lead to a variety of congenital anemias including beta thalassemia, hereditary pyropoikilocytosis, hereditary elliptocytosis, and hereditary spherocytosis. Aberrant mRNA splicing has subsequently been shown to lead to acquired anemias in subsets of patients with myelodysplastic syndromes (MDS). Somatic missense mutations in components of the spliceosome are the most common category of mutations in MDS. These point mutations lead to changes in the RNA binding specificity of the involved proteins and aberrant splicing of a subset of transcripts. Mutant SF3B1, the most commonly mutated splicing factor in MDS, has been shown to cause aberrant pre-mRNA splicing and an increase in transcripts predicted to undergo NMD due to use of upstream, cryptic 3' splice sites. Our group and others evaluating the strong genotype-phenotype association between SF3B1 point mutations and subtypes of MDS with ring sideroblasts have shown that the expression of the mitochondrial iron transporter, ABCB7, is decreased in samples from SF3B1-mutant MDS patients due to cryptic 3' splice site selection and introduction of a PTC between exons 8 and 9. The identification and functional validation of additional aberrantly spliced mutant-SF3B1 target genes is ongoing, with the goal of understanding how point mutations in a core component of the mRNA splicing machinery can lead to such specific effects on erythroid maturation. Disclosures No relevant conflicts of interest to declare.

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Splice site mutations are a common cause of X-linked chronic granulomatous disease

Blood ◽

10.1182/blood.v80.6.1553.bloodjournal8061553 ◽

1992 ◽

Vol 80 (6) ◽

pp. 1553-1558 ◽

Cited By ~ 1

Author(s):

M de Boer ◽

BG Bolscher ◽

MC Dinauer ◽

SH Orkin ◽

CI Smith ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

Splice Site ◽

Messenger Rna ◽

Mrna Splicing ◽

Molecular Defect ◽

Granulomatous Disease ◽

Splice Sites ◽

Nucleotide Substitutions ◽

Common Cause ◽

Donor Splice

Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp91-phox). We studied the molecular defect in four patients with X- linked CGD. In a fifth family, we studied the mother of a patient with X-linked CGD who had died before our investigations. Gp91-phox messenger RNA (mRNA) was reverse transcribed into cDNA and the coding region was amplified by polymerase chain reaction into three fragments. Sequence analysis showed the absence of the exon 7, 5, 3, and 2 sequences in patients 1, 2, 3, and 4, respectively. In carrier 5, we found both normal cDNA and cDNA that lacked 57 3′-nucleotides of exon 6. We analyzed the splice sites of the flanking introns of the missing exons. In patients 1, 2, and 3, we found single nucleotide substitutions within the first five positions of the down-stream 5′ donor splice sites. In patient 4, a similar substitution was found at position -1 of the 3′ acceptor splice site of intron 1. In carrier 5, no mutation was found in the exon 6-intron 6 boundary sequence. Instead, a single substitution was observed in exon 6 (C----A at nucleotide 633) that created a new donor splice site. Apparently, mRNA splicing occurs preferentially at this newly created splice site. We conclude that the absence of the exon sequences in the gp91-phox mRNA of these patients is due to splicing errors. Of 30 European X-linked CGD patients studied by us so far, five appear to be caused by mutations that affect correct mRNA splicing. Thus, such mutations appear to be a common cause of X-linked CGD.

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Pan-cancer repository of validated natural and cryptic mRNA splicing mutations

F1000Research ◽

10.12688/f1000research.17204.2 ◽

2019 ◽

Vol 7 ◽

pp. 1908 ◽

Cited By ~ 1

Author(s):

Ben C. Shirley ◽

Eliseos J. Mucaki ◽

Peter K. Rogan

Keyword(s):

Splice Junction ◽

Cancer Genome ◽

Mrna Splicing ◽

The Cancer Genome Atlas ◽

Splice Sites ◽

Global Alliance ◽

Multiple Tumor ◽

Link Type ◽

Splicing Mutations ◽

Single Nucleotide Polymorphism Database

We present a major public resource of mRNA splicing mutations validated according to multiple lines of evidence of abnormal gene expression. Likely mutations present in all tumor types reported in the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) were identified based on the comparative strengths of splice sites in tumor versus normal genomes, and then validated by respectively comparing counts of splice junction spanning and abundance of transcript reads in RNA-Seq data from matched tissues and tumors lacking these mutations. The comprehensive resource features 341,486 of these validated mutations, the majority of which (69.9%) are not present in the Single Nucleotide Polymorphism Database (dbSNP 150). There are 131,347 unique mutations which weaken or abolish natural splice sites, and 222,071 mutations which strengthen cryptic splice sites (11,932 affect both simultaneously). 28,812 novel or rare flagged variants (with <1% population frequency in dbSNP) were observed in multiple tumor tissue types. Single variants or chromosome ranges can be queried using a Global Alliance for Genomics and Health (GA4GH)-compliant, web-based Beacon “Validated Splicing Mutations” either separately or in aggregate alongside other Beacons through the public Beacon Network, as well as through our website.

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