Real-time visualization of spliceosome assembly reveals basic principles of splice site selection

The spliceosome is a megadalton protein-RNA complex which removes introns from pre-mRNA, yet the dynamic early assembly steps have not been structurally resolved. Specifically, how the spliceosome selects the correct 3' splice site (3'SS) amongst highly similar non-functional sites is not known. Here, we develop a kinetic model of splice site selection based on single-molecule U2AF heterodimer imaging in vitro and in vivo. The model successfully predicts alternative splicing patterns and indicates that 3'SS selection occurs while U2AF is in complex with the spliceosome, not during initial binding. This finding indicates the spliceosome operates in a 'partial' kinetic proofreading regime, catalyzed in part by the helicase DDX42, which increases selectivity to the underlying U2AF binding site while still allowing for efficient forward progression. Overall design: To investigate the relationship betwen relative binding site strength and 3' splice site selection, we performed RNA sequencing in human bronchial epithial cells (HBECs) under two conditions (a) transfection with control siRNA and (b) transfection with DDX42 siRNA. We then performed transcript quantification and alternative splicing analysis using data obtained from RNA-seq of siControl and siDDX42 samples.