Noncanonical GC-AG and AT-AC sequences at the splice sites occur in 0.56 and 0.09% of the splice site pairs. In most of cases (98.7%), the exon/intron boundary sequences contain GT and AG motifs at the 5′ and 3′ ends of the intron, respectively. In this complex, two transesterification reactions take place, intron is removed, and ends of exon are joined (Fredericks et al. Further action of additional RNA helicases leads to change of spliceosome conformation that leads to the release of U1 and U4 snRNPs, the interaction between U6 with U2 snRNP, and the formation of a pre-mRNA loop and the C complex. The interaction between the branch point and the U2snRNP protein is stabilized by specific RNA helicases (Prp5 and Sub2), and this is a signal for the recruitment of U4/5/6 tri-snRNP and formation of the B complex (pre-catalytic spliceosome). Then, the SF1 is displaced from the branch point by the U2 snRNP, and the ATP-dependent (A) complex is formed. The SF1 is recognized and bound by the U2AF65 protein that also binds to the polypyrimidine sequence located between the branch point and 3′ end of the intron. In the same time, the SF1 protein binds to the branch point. The U1 snRNP recognizes and binds to the complementary AG-GU sequence at the donor splice site (5′ end of the intron). During the splicing process, four complexes between the pre-mRNA and spliceosome are formed. The first step is the recognition of the splicing sites at intron/exon junctions, and the second one is the intron removal and exon ends joining. The splicing process is performed in two steps. This article summarizes the current knowledge about the “splicing mutations” and methods that help to identify such changes in clinical diagnosis. However, it should be underlined that the results of such tests are only predictive, and the exact effect of the specific mutation should be verified in functional studies. The bioinformatic algorithms can be applied as a tool to assess the possible effect of the identified changes. The application of modern techniques allowed to identify synonymous and nonsynonymous variants as well as deep intronic mutations that affected pre-mRNA splicing. Recent research has underlined the abundance and importance of splicing mutations in the etiology of inherited diseases. Usually such mutations result in errors during the splicing process and may lead to improper intron removal and thus cause alterations of the open reading frame. The splicing mutation may occur in both introns and exons and disrupt existing splice sites or splicing regulatory sequences (intronic and exonic splicing silencers and enhancers), create new ones, or activate the cryptic ones. Point mutations at these consensus sequences can cause improper exon and intron recognition and may result in the formation of an aberrant transcript of the mutated gene. Precise pre-mRNA splicing, essential for appropriate protein translation, depends on the presence of consensus “cis” sequences that define exon-intron boundaries and regulatory sequences recognized by splicing machinery.
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