Compensatory signals associated with the activation of human GC 5′ splice sites

Abstract

GC 5′ splice sites (5′ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5′ss activated by a mutation in BTK intron 3. This GC 5′ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5′ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5′ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5′ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5′ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5′ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5′ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites.

CYCLIN-DEPENDENT KINASE G1 Is Associated with the Spliceosome to RegulateCALLOSE SYNTHASE5Splicing and Pollen Wall Formation inArabidopsis

“…For this rare type of intron, various SR proteins bind to the U1 snRNP-pre-mRNA complex to stabilize the base pairing (Black, 2003). Human SR proteins 9G8 and SC35 can promote the splicing of introns with the GC 59 SS (Kralovicova et al, 2011). In cdkg1, the low splicing efficiency of the CalS5 intron 6 ( Figure 3D) verifies the weakness of this GC 59 SS.…”

“…A GC 59 SS is intrinsically weaker than the major GT 59 SS because the T>C substitution introduces a mismatch in the base pairing between the splice site and U1 snRNA (Kralovicova et al, 2011). For this rare type of intron, various SR proteins bind to the U1 snRNP-pre-mRNA complex to stabilize the base pairing (Black, 2003).…”

“…This facilitates spliceosome removal of this rare type of intron. Kralovicova et al (2011) reported that the flanking sequences could enhance the splicing efficiency of an intron with the GC 59 SS. Therefore, both CDKG1 and the flanking sequences are important for efficient splicing of the introns with a GC 59 SS.…”

“…These splicing factors also include some SR protein family members, such as 9G8 and SC35. They are essential for the recognition of suboptimal splice sites (Kralovicova et al, 2011). Therefore, SR proteins are important early in spliceosome assembly (Shepard and Hertel, 2009).…”

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

“…This mechanism has been observed in multiple diseases, including cystic fibrosis (22) and ataxia telangiectasia (23,24) among others. In XLA, we have previously identified and described 2 such families (25,26).…”