Mutational analysis of splicing activation by the USSE motif in the U11/U12-specific 65K gene

Splicing of introns and joining of exons to yield mature and functional mRNA molecules is carried out through two sequential trans-esterification reactions by two distinct spliceosomes; the major U2-dependent and the minor U12-dependent spliceosomes. Alternative splicing is common in eukaryotes and occurs mostly with U2 type introns. The level of minor spliceosomes specific proteins, U11-48K and U11/U12-65K, are regulated via alternative splicing where U11 snRNP binds to the USSE (U11 snRNP binding splicing enhancer), a tandem repeat of 5’ss of U12-type introns, and activates an upstream 3’ss of the U2 dependent spliceosome. Here, I carried forward the study done in Verbeeren et al, 2010, through manipulation of the distance between the two 5’ss within the USSE element, and the distance between the upstream 3’splice site and USSE. The mechanisms through which USSE recognition by the U11 snRNP is achieved, as well as the distance constraints the USSE element imposes on alternative splicing, were analyzed. RT-PCR analysis shows that (a) occurrence of the alternative splicing event drops with reduced distance between 3’ss-USSE, and is finally lost when the distance is reduced to zero, (b) both increased and decreased distance between the two 5’ss within the USSE resulted in reduced long isoform formation. The data set suggests that the simultaneous binding of two 5’ss within USSE by U11/U12 snRNPs and their interaction is necessary for USSE mediated alternative splicing activation. So, the evolutionary conserved sequence within USSE, and the distance to 3’ss are critical for USSE mediated alternative splicing