An Allosteric Network for Spliceosome Activation Revealed by High-Throughput Suppressor Analysis in .

Selection of suppressor mutations that correct growth defects caused by substitutions in an RNA or protein can reveal functionally important molecular structures and interactions in living cells. This approach is particularly useful for the study of complex biological pathways involving many macromolecules, such as premessenger RNA (pre-mRNA) splicing. When a sufficiently large number of suppressor mutations is obtained and structural information is available, it is possible to generate detailed models of molecular function. However, the laborious and expensive task of identifying suppressor mutations in whole-genome selections limits the utility of this approach. Here I show that a custom targeted sequencing panel can greatly accelerate the identification of suppressor mutations in the genome. Using a panel that targets 112 genes encoding pre-mRNA splicing factors, I identified 27 unique mutations in six protein-coding genes that each overcome the cold-sensitive block to spliceosome activation caused by a substitution in U4 small nuclear RNA. When mapped to existing structures of spliceosomal complexes, the identified suppressors implicate specific molecular contacts between the proteins Brr2, Prp6, Prp8, Prp31, Sad1, and Snu114 as functionally important in an early step of catalytic activation of the spliceosome. This approach shows great promise for elucidating the allosteric cascade of molecular interactions that direct accurate and efficient pre-mRNA splicing and should be broadly useful for understanding the dynamics of other complex biological assemblies or pathways.