Exon, intron and splice site locations in the spliceosomal B complex.
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ABSTRACT:
SUBMITTER: Wolf E
PROVIDER: S-EPMC2726701 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Spliceosomal twin introns (stwintrons) are introns where any of the three consensus sequences involved in splicing is interrupted by another intron (internal intron). In Aspergillus nidulans, a donor-disrupted stwintron (intron-1) is extant in the transcript encoding a reticulon-like protein. The orthologous transcript of Aspergillus niger can be alternatively spliced; the exon downstream the stwintron could be skipped by excising a sequence that comprises this stwintron, the neighbouring intron-2, and the exon bounded by these. This process involves the use of alternative 3' splice sites for the internal intron, the resulting alternative intervening sequence being a longer 3'-extended stwintron. In 29 species of Onygenales, a multi-step splicing process occurs in the orthologous transcript, in which a complex intervening sequence including the stwintron and neigbouring intron-2, generates by three splicing reactions a "second order intron" which must then be excised with a fourth splicing event. The gene model in two species can be envisaged as one canonical intron (intron-1) evolved from this complex intervening sequence of nested canonical introns found elsewhere in Onygenales. Postulated splicing intermediates were experimentally verified in one or more species. This work illustrates a role of stwintrons in both alternative splicing and the evolution of intron structure.
| S-EPMC6616335 | biostudies-literature
Project description:A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.
| S-EPMC3923125 | biostudies-literature
Project description:A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.
| S-EPMC8706089 | biostudies-literature
Project description:Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5' and 3' splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5' and 3' splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.
| S-EPMC9225289 | biostudies-literature
Project description: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 of the 3'YAG consensus, were more frequent in exons than in introns. They clustered in approximately 20 nt region adjacent to authentic 3'ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3'ss that were induced by mutations outside the 3'YAG consensus (designated 'de novo') were in introns. The activation of intronic de novo 3'ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3'ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro-Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3'ss. Finally, AG-creating mutations in the PPT that produced aberrant 3'ss upstream of the predicted BPS in vivo shared a similar 'BPS-new AG' distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3'ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.
| S-EPMC1197134 | biostudies-literature
Project description:Acceptor splice site recognition (3' splice site: 3'ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3'ss, of which U2AF35 has a dual function: (i) It binds to the intron-exon border of some 3'ss and (ii) mediates enhancer-binding splicing activators' interactions with the spliceosome. Alternative mechanisms for 3'ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3'ss recognition where the intron-exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3'ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3'ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons' inclusion. Most probably, both factors stochastically bind the 3'ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3'ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants' effects on splicing.
| S-EPMC7554774 | biostudies-literature
Project description:Germline TP53 splicing variants are uncommon, and their clinical relevance is unknown. However, splice-altering variants at exon 4-intron 4 junctions are relatively enriched in pediatric adrenocortical tumors (ACT). Nevertheless, family histories of cancer compatible with classic Li-Fraumeni syndrome are rarely seen in these patients. We used conventional and in silico assays to determine protein stability, splicing, and transcriptional activity of 10 TP53 variants at exon 4-intron 4 junctions and analyzed their clinical correlates. We reviewed public databases that report the impact of TP53 variants in human cancer and examined individual reports, focusing on family history of cancer. TP53 exon 4-intron 4 junction germline variants were identified in 9 of 75 pediatric ACTs enrolled in the International Pediatric Adrenocortical Tumor Registry and Children's Oncology Group ARAR0332 study. An additional eight independent TP53 variants involving exon 4 splicing were identified in the Pediatric Cancer Genome Project (n = 5,213). These variants resulted in improper expression due to ineffective splicing, protein instability, altered subcellular localization, and loss of function. Clinical case review of carriers of TP53 exon 4-intron 4 junction variants revealed a high incidence of pediatric ACTs and atypical tumor types not consistent with classic Li-Fraumeni syndrome. Germline variants involving TP53 exon 4-intron 4 junctions are frequent in ACT and rare in other pediatric tumors. The collective impact of these germline TP53 variants on the fidelity of splicing, protein structure, and function must be considered in evaluating cancer susceptibility. IMPLICATIONS: Taken together, the data indicate that splice variants at TP53 codon 125 and surrounding bases differentially impacted p53 gene expression and function.
| S-EPMC8816887 | biostudies-literature
Project description:Short introns occur in numerous protist lineages, but there are no reports of intervening sequences in the protists Giardia lamblia and Trichomonas vaginalis, which may represent the deepest known branches in the eukaryotic line of descent. We have discovered a 35-bp spliceosomal intron in a gene encoding a putative [2Fe-2S] ferredoxin of G. lamblia. The Giardia intron contains a canonical splice site at its 3' end (AG), a noncanonical splice site at its 5' end (CT), and a branch point sequence that fits the yeast consensus sequence of TACTAAC except for the first nucleotide (AACTAAC). We have also identified several G. lamblia genes with spliceosomal peptides, including homologues of eukaryote-specific spliceosomal peptides (Prp8 and Prp11), several DExH-box RNA-helicases that have homologues in eubacteria, but serve essential functions in the splicing of introns in eukaryotes, and 11 predicted archaebacteria-like Sm and like-Sm core peptides, which coat small nuclear RNAs. Phylogenetic analyses show the Giardia Sm core peptides are the products of multiple, ancestral gene duplications followed by divergence, but they retain strong similarity to Sm and like-Sm peptides of other eukaryotes. Although we have documented only a single intron in Giardia, it likely has other introns and fully functional, spliceosomal machinery. If introns were added during eukaryotic evolution (the introns-late hypothesis), then these results push back the date of this event before the branching of G. lamblia.
| S-EPMC122587 | biostudies-literature
Project description:U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.
| S-EPMC86782 | biostudies-literature
Project description:Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.
| S-EPMC9984181 | biostudies-literature