Poster abstracts
Poster number 116 submitted by Chad Ratterman
Branchpoint identity and branch length effects on lariat debranching enzyme activity
Timothy Chad Ratterman (Chemistry, Carnegie Mellon University, Center for Nucleic Acids Science and Technology), Subha R. Das (Chemistry, Carnegie Mellon University, Center for Nucleic Acids Science and Technology)
Abstract:
Lariat introns are formed during splicing when the 5ʹ-end of the intron is attacked by the 2ʹ-hydroxyl of an internal adenosine residue. Consequently, the canonical branchpoint residue in an excised lariat intron is an adenosine that contains a 2ʹ,5ʹ-phosphodiester linkage. These lariat introns must subsequently be opened at the 2ʹ,5ʹ-linkage by the lariat debranching enzyme (Dbr1p) before they can continue on in other regulatory processes. Hence the mechanism of Dbr1p binding and cleavage of the 2',5'-linkage at the branchpoint is of significant interest. Previous work from our lab has shown that S. cerevisiae and E. histolytica Dbr1p are capable of cleaving backbone branched RNAs (bbRNAs) containing non-adenosine branchpoint residues. Further, a recently published crystal structure of Dbr1p shows the nucleotide arms bound by the enzyme differ in the number of bound residues. Here we detail our analysis of the kinetics of binding and cleavage activity of E. histolytica Dbr1p using both bbRNAs substrates that contain non-canonical branchpoint residues and branched RNAs with a different number of residues in the nucleotide arms. These data provide new details on Dbr1p interactions at and close to the branchpoint of lariat introns.
Keywords: Lariat Debranching Enzyme, Branched RNAs, Lariat Introns