Interplay of primary sequence and RNA secondary structure in determining 5′ splice site choice
Selective use of 5′ splice sites is a common mechanism by which pre-mRNAs are alternatively spliced. Whereas the sequence requirements of 5′ splice site choice have been well characterized, other important determinants remain poorly defined. Here we apply a combination of structural mapping by SHAPE-MaP and targeted mutational analysis in a cell-based system to comprehensively probe the interplay of primary sequence, secondary RNA structure, regulatory elements and linear splice site position to determine mechanisms of splice site choice in vivo. Using the disease-causing alternative 5′ splice site selection in LMNA in the premature aging disorder Hutchinson-Gilford Progeria Syndrome as a model system, we identify RNA secondary structural elements near the alternative 5′ splice sites. We show that splice site choice is significantly influenced by the structural context of the available splice sites. While local structure alone is not sufficient to account for splice site selection, the choice of 5′ splice sites depends on the structural stability of the 5′ splice site region which is conferred by downstream elements. In addition, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5′ splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results reveal an intricate interplay amongst RNA sequence, secondary structure and splice site position in determining 5′ splice site choice.