Despite a growing number of splicing mutations found in hereditary diseases, utilization aberrant splice sites and their effects on gene expression remain challenging to predict. We compiled sequences 346 5′splice (5′ss) that were activated by 166 human disease genes. Mutations within the 5′ss consensus accounted for 254 cryptic elsewhere 92 de novo 5′ss. Point leading activation most common first intron nucleotide, followed fifth nucleotide. Substitutions at position +5 exclusively G>A...

Missense, nonsense, and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency among disease-causing exonic substitutions is unknown. Here, we have tested missense deposited BRCA1 mutation databases unclassified variants for effects on exon inclusion. Analysis 21 using minigene assays revealed a single exon-skipping c.231G>T. Comprehensive mutagenesis an adjacent 12-nt segment showed that this resulted higher level...

Abstract Selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID) are the most primary immunodeficiencies in humans. A high degree of familial clustering, marked differences population prevalence among ethnic groups, association IgAD CVID families, a predominant inheritance pattern multiple-case pedigrees have suggested strong, shared genetic predisposition. Previous linkage, case-control, family-based studies mapped an IgAD/CVID susceptibility locus, designated IGAD1, to...

Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance selection mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic aberrant were activated by splice-site mutations human disease genes have lower frequencies enhancers higher silencers than average exons. Conversely, intronic more less introns. Exons skipped as result smaller, had...

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. deficiency (PD) causes developmental delay coupled intellectual disability spinal, cardiac, ocular renal defects, but PD pathogenesis not understood. Using RNA-Seq, we identify human PUF60-regulated exons show preferentially acts as their activator. PUF60-activated internal are enriched for Us upstream 3′ splice sites (3′ss), preceded...

We show that the allele-dependent expression of transcripts encoding soluble HLA-DQβ chains is determined by branchpoint sequence (BPS) haplotypes in DQB1 intron 3. BPS RNAs associated with low inclusion transmembrane exon mature showed impaired binding to splicing factor 1 (SF1), indicating alternative controlled differential recognition early during spliceosome assembly. also demonstrate naturally occurring human point mutations alter and lead recognizable phenotypes cluster BP position −2...

We compiled sequences of previously published aberrant 3′ splice sites (3′ss) that were generated by mutations in human disease genes. Cryptic 3′ss, defined here as those resulting from a mutation the 3′YAG consensus, more frequent exons than introns. They clustered ∼20 nt region adjacent to authentic suggesting their under-representation introns is due depletion AG dinucleotides polypyrimidine tract (PPT). In contrast, most 3′ss induced outside consensus (designated ' de novo ') The...

Predisposition to type 1 diabetes and juvenile obesity is influenced by the susceptibility locus IDDM2 that includes insulin gene (INS). Although risk conferred has been attributed a minisatellite upstream of INS, intragenic variants have not ruled out. We examined whether INS polymorphisms affect pre-mRNA splicing proinsulin secretion using minigene reporter assays. show IVS1-6A/T (−23HphI+/−) key variant influences alternative intron through differential recognition its 3′ splice site. The...

The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) AG dinucleotides at 3′ splice site (3′ss). gene encoding U2AF35 (U2AF1) alternatively spliced, giving rise to two isoforms U2AF35a U2AF35b. Here, we knocked down each isoform characterized transcriptomes HEK293 cells with varying U2AF35/U2AF65 U2AF35a/b ratios. Depletion both preferentially modified alternative processing events without widespread...

GC 5' splice sites (5'ss) are present in ∼1% of human introns, but factors promoting their efficient selection poorly understood. Here, we describe a case X-linked agammaglobulinemia resulting from 5'ss activated by mutation BTK intron 3. This was intrinsically weak, yet it selected >90% primary transcripts the presence strong and intact natural GT counterpart. We show that this required high density GAA/CAA-containing splicing enhancers exonized segment promoted SR proteins 9G8, Tra2β SC35....

The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether major components implicated selection, proteins U2AF35 and U2AF65 U2 snRNP, able recognize alternative candidate or restricted one pre-specified site. In presence adenosine triphosphate (ATP), all three bind a 1:1 stoichiometry with 3΄ss. Pre-mRNA molecules two 3΄ss can be bound concurrently by...

The branch point sequence (BPS) is a conserved splicing signal important for spliceosome assembly and lariat intron formation. BPS mutations may result in aberrant pre-mRNA genetic disorders, but their phenotypic consequences have been difficult to predict, largely due highly degenerate nature of the consensus. Here, we examined pattern nine reporter pre-mRNAs that previously shown give rise human hereditary diseases as single-nucleotide substitutions predicted BPS. Increased exon skipping...

Cryptic exons or pseudoexons are typically activated by point mutations that create GT AG dinucleotides of new 5′ 3′ splice sites in introns, often repetitive elements. Here we describe two cases tetrahydrobiopterin deficiency caused improving the branch sequence and polypyrimidine tracts repeat-containing PTS gene. In first case, demonstrate a novel pathway antisense Alu exonization, resulting from an intronic deletion removed poly(T)-tail AluSq. The brought favorable within proximity...

Transcriptomic diversity in primates was considerably expanded by exonizations of intronic Alu elements. To better understand their cellular mechanisms we have used structure-based mutagenesis coupled with functional and proteomic assays to study the impact successive primate mutations combinations on inclusion a sense-oriented AluJ exon human F8 gene. We show that splicing outcome predicted consecutive RNA conformation changes than computationally derived regulatory motifs. also demonstrate...

Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns increase splicing-mediated gene expression not developed. Here we show reduction INS intron 1 retention by SSOs bind transcripts derived from a human haplotype expressing low levels proinsulin. This is tagged polypyrimidine tract variant rs689 decreases the efficiency splicing and increases relative abundance mRNAs with extended 5' untranslated...

Splice-site selection is controlled by secondary structure through sequestration or approximation of splicing signals in primary transcripts but the exact role even simplest and most prevalent structural motifs exon recognition remains poorly understood. Here we took advantage a single-hairpin that was activated mammalian-wide interspersed repeat (MIR) mutation stabilizing terminal triloop, with splice sites positioned close to each other lower stem hairpin. We first show MIR inclusion mRNA...

Abstract Alternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression soluble DQβ. We have recently shown that differential inclusion this mature transcripts largely due to intron 3 variants the branch point sequence (BPS) polypyrimidine tract. To identify additional regulatory cis-elements contribute haplotype-specific DQB1, we systematically examined effect guanosine (G) repeats on removal. found GGG or GGGG generally improved DQB1 3, except for those were...

Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin gene (INS) and differential steady-state levels of INS RNA from T1D-predisposing -protective haplotypes. Here, we show that haplotype-specific expression is determined by variants control splicing efficiency intron 1. The adenine allele at IVS1-6 (rs689), which rapidly expanded in modern humans, renders 3' splice site this more dependent auxiliary factor U2 small nuclear...

The auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation lariat introns. gene for the 35-kD subunit U2AF gives rise to two protein isoforms (termed U2AF35a U2AF35b) that are encoded by alternatively spliced exons 3 Ab, respectively. splicing sequences exon less favorable than yet expression is higher U2AF35b across tissues. We show repression facilitated weak, closely spaced BPs next a long polypyrimidine tract Ab. Each BP...

Abstract Ca2+-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH ATP supply by mutually exclusive OGDH exons 4a 4b. Here we show that their splicing is enforced distant lariat branch points (dBPs) located near 5′ splice site intervening intron. dBPs restrict intron length prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed a single dominant in anamniotes...

Transposed elements (TEs) have dramatically shaped evolution of the exon-intron structure and significantly contributed to morbidity, but how recent TE invasions into older TEs cooperate in generating new coding sequences is poorly understood. Employing an updated repository boundaries induced by pathogenic mutations, termed DBASS, here we identify novel clusters that facilitated exon selection. To explore extent which such exons maintain RNA secondary their progenitors, carried out...