Synthesis and Evaluation of Non-Canonical Substrates for Lariat Debranching Enzyme

Lariat debranching enzyme (Dbr1p), which hydrolyzes 2ʹ,5ʹ-phosphodiester bonds in lariat introns produced during pre-mRNA splicing, is highly conserved across organisms. This cleavage activity is the first step in the processing of introns into regulatory RNAs. Despite sharing substantial structural similarity with other metallophosphoesterases, Dbr1p shows significantly greater specificity than other members of this superfamily. Because of this specificity and the high uniformity of its cellular substrate the canonical Dbr1p substrate can be tightly defined as a lariat intron arising from the splicing of precursor messenger RNA (pre-mRNA).

While the canonical substrate can be tightly defined, Dbr1p has shown significant substrate flexibility in biochemical assays. Investigation of the Dbr1p processing of non-canonical substrates was performed and expands our knowledge of enzyme active substrates. The highly conserved adenine nucleobase on the branchpoint residue can be substituted with the other standard RNA nucleobases without abolishing enzyme activity. Real-time kinetics assays show that changes in the hydrogen bond donor/acceptor pattern play a larger role in substrate binding than does nucleobase ring size. Mutational study of putative contacts between the enzyme and branchpoint nucleobase identify redundancy in substrate binding allowing for continued enzyme function.

A circular single-stranded small interfering RNA mimic of microRNA is synthesized. Previous attempts to prepare lariat mimics of microRNA were hindered by complicated synthesis protocols and low yields. The circular RNA prodrug is linearized by Dbr1p-mediated cleavage of an internal 2ʹ,5ʹ-phosphodiester bond to produce the active form. Once demonstrated to be active in the RNA interference pathway within the cell, this miRNA mimic could be used therapeutically in much the same manner as standard small interfering RNA.

A proof-of-concept synthesis is performed to synthesize a single RNA bearing multiple branches. This substrate is fluorescently labeled to allow for visualization of the cleavage products following incubation with Dbr1p. This assay could help elucidate the mechanism by which the enzyme locates the branchpoint.

Upstream of Dbr1p action is splicing of pre-mRNA which is catalyzed by the spliceosome. The catalytic core of the spliceosome is composed of RNAs which interact with the intron. With synthetic access to branched RNAs, we are able to model the spliceosome core both before and after the branching step of splicing. As the spliceosomal RNA is not entirely duplexed, we prepared truncated constructs to mimic the observed interactions. Melting temperature analysis is used to investigate the strength of the RNA interactions within the spliceosome. Biologically relevant duplexes of the spliceosome core before and after the first splicing reaction are prepared and their stability measured to determine the stabilizing or destabilizing effect of the branch. Pseudouridine residues are included to elucidate the magnitude of their stabilizing effect upon the duplexes.

This thesis focuses on the synthesis and evaluation of non-canonical substrates for Dbr1p to better understand their binding and turnover by the enzyme.