Reprogramming Msl5 to recognize mutant branch point sequences during spliceosome assembly

Abstract

Saccharomyces cerevisiae Msl5 is an essential protein responsible for the assembly of a catalytically active spliceosome. Msl5 mediates spliceosome assembly through the recognition of the intronic branchpoint sequence 5′‐UACUAAC. Upon binding, Msl5 establishes an interaction with U1 bound to the 5′ splice site before recruiting the U2 snRNP to the intronic branchpoint sequence. We have generated and expressed mutant Msl5 proteins containing L176N and L167A/Q255A mutations, designed to bind specifically to mutant branch point sequences 5′‐UACUACC and 5′‐UACAAAC, respectively. Electrophoretic mobility shift assays will be performed to determine if reprogrammed Msl5 can specifically recognize and bind to a mutant branch point sequence. The reprogramming of Msl5 can be used to generate an orthogonal Msl5 system in vivo, and to analyze the effectiveness of intron splicing containing different branchpoint sequences.