Investigation of Functionally Critical Residues in the Pseudo‐ Helicase Domain of Brr2

Abstract

Pre‐mRNA splicing is the removal of introns from mRNA to create mature mRNA. The conformational rearrangements that are required to remove introns are driven by a family of helicases called DExD/H box proteins. A member of this family of helicases, Brr2, is an RNA dependent ATPase that unwinds U4/U6 prior to catalytic activation of the spliceosome and unwinds U2/U6 during disassembly. Brr2 has a unique structure composed of an N‐terminal domain and two modules that resemble the DNA Helicase Hel308. Previous work identified the Hel308‐I domain of Brr2 contains the DEIH motif and is responsible for Brr2's helicase activity. The Hel308‐II domain of Brr2 lacks the canonical DExD/H motif and is not directly responsible for RNA unwinding. However, the domain is conserved, implying the domain's importance to the protein. Using Saccharomyces cerevisiae, we have identified cold‐sensitive and temperature‐sensitive mutations in the Hel308‐II domain, including several mutations that cluster around the putative ATP binding pocket. These findings support a functional importance of ATP binding to this region. We will present data on the ATP binding properties of these mutations and their effects on in vitro splicing.