Abstract
Mutations affecting constitutive splice donor sites (5'ss) are among the most frequent genetic defects that disrupt the normal splicing process. Pre-mRNA splicing requires the correct identification of a number of cis-acting elements in an ordered fashion. By disrupting the complementarity of the 5'ss with the endogenous small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, 5'ss mutations may result in exon skipping, intron retention or activation of cryptic splice sites. Engineered modification of the U1 snRNA seemed to be a logical method to overcome the effect of those mutations. In fact, over the last years, a number of in vitro studies on the use of those modified U1 snRNAs to correct a variety of splicing defects have demonstrated the feasibility of this approach. Furthermore, recent reports on its applicability in vivo are adding up to the principle that engineered modification of U1 snRNAs represents a valuable approach and prompting further studies to demonstrate the clinical translatability of this strategy.Here, we outline the design and generation of U1 snRNAs with different degrees of complementarity to mutated 5'ss. Using the HGSNAT gene as an example, we describe the methods for a proper evaluation of their efficacy in vitro, taking advantage of our experience to share a number of tips on how to design U1 snRNA molecules for splicing rescue.
Highlights
Using the HGSNAT gene as an example, we describe the methods for a proper evaluation of their efficacy in vitro, taking advantage of our experience to share a number of tips on how to design U1 snRNA molecules for splicing rescue
The U1 small nuclear ribonucleoprotein (U1 snRNP) is a key molecule involved in an early event of the splicing process
Like other snRNPs involved in the overall splicing regulation process, it contains a small RNA complexed with several proteins, namely seven Smith antigen (Sm) proteins and three U1-specific proteins (U1A, U1C, and U170K) [1]
Summary
The U1 small nuclear ribonucleoprotein (U1 snRNP) is a key molecule involved in an early event of the splicing process. A straightforward prediction of the effect of mutations flanking the canonical GU site without a direct assessment of the mature mRNA produced can be quite challenging It is the variable degree of degeneration of 50ss and the surprising heterogeneity existing among human spliceosomal snRNA, which allows for splicing correction using modified exogenous U1 snRNAs. Overall, the rationale on the use of modified U1 snRNAs to correct splicing defects is as simple as it can be: as 50ss mutations alter the 50ss recognition by the endogenous U1 snRNA, exogenous U1 snRNAs may be engineered through complementary base pairing in order to correctly recognize the mutated allele and initiate spliceosome assembly, suppressing the mutation effect. We elaborate on the relevance of further addressing the treatment’s effect at protein level
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