Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition.

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.

SERPING1 exon 3 splicing variants using alternative acceptor splice sites.

Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3' splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3' ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3' ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3' ss seems to be co-regulated with the authentic 3' ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.

Systematic analysis of splicing defects in selected primary immunodeficiencies-related genes.

Both variants affecting splice sites and those in splicing regulatory elements (SREs) can impair pre-mRNA splicing, eventually leading to severe diseases. Despite the availability of many prediction tools, prognosis of splicing affection is not trivial, especially when SREs are involved. Here, we present data on 92 in silico-/55 minigene-analysed variants detected in genes responsible for the primary immunodeficiencies development (namely BTK, CD40LG, IL2RG, SERPING1, STAT3, and WAS). Of 20 splicing-affecting variants, 16 affected splice site while 4 disrupted potential SRE. The presence or absence of splicing defects was confirmed in 30 of 32 blood-derived patients' RNAs. Testing prediction tools performance, splice site disruptions and creations were reliably predicted in contrast to SRE-affecting variants for which just ESRseq, ΔHZEI-scores and EX-SKIP predictions showed promising results. Next, we found an interesting pattern in cryptic splice site predictions. These results might help PID-diagnosticians and geneticists cope with potential splicing-affecting variants.

Exon first nucleotide mutations in splicing: evaluation of in silico prediction tools.

Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.

Myocardial injury is decreased by late remote ischaemic preconditioning and aggravated by tramadol in patients undergoing cardiac surgery: a randomised controlled trial.

The purpose of this study was to test, whether the late phase of remote ischaemic preconditioning (L-RIPC) improves myocardial protection in coronary artery bypass grafting (CABG) with cold-crystalloid cardioplegia and whether preoperative tramadol modifies myocardial ischaemia-reperfusion injury using the same group of patients in a single-blinded randomized controlled study. One hundred and one adult patients were randomly assigned to either the L-RIPC, control or tramadol group. L-RIPC consisted of three five-minute cycles of upper limb ischaemia and three five-minute pauses using blood pressure cuff inflation 18 hours prior to the operation. Patients in the tramadol group received 200 mg tramadol retard at 19:00 hours, the day before the operation and at 06:00 hours. Serum troponin I levels were measured at eight, 16 and 24 hours after surgery. Myocardial samples for inducible and endothelial nitric oxide synthases (iNOS, eNOS) estimation were drawn twice: before and after cannulation for cardiopulmonary bypass from the auricle of the right atrium. We found that L-RIPC can reduce injury beyond the myocardial protection provided by cold-crystalloid cardioplegia, and tramadol worsened myocardial injury after CABG. Expressions of iNOS were increased in the control (significantly) and L-RIPC groups and dampened in the tramadol group.