Biochemical Investigation of Lariat Debranching Enzyme and Spliceosomal RNA through the Use of Backbone Branched RNA
Creators: name: Mack, Stephanie
Publisher: name: Carnegie Mellon University
Year: 2019
Subjects: subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences

description: Lariat introns are generated during pre-mRNA splicing. The 2'-OH of the branch point residue is linked to the 5'-terminus of the intron through a 2'-5'-phosphodiester bond. The 2'-5'-<br>phosphodiester bond is not processed by traditional exo or endonucleases and is only cleaved by lariat debranching enzyme (Dbr1p). Introns are involved in a variety of regulatory processes in the cell and thus, Dbr1p is upstream from many critical cellular functions. Dbr1p is structurally<br>very similar to other non-specific metallophosphoesterases and it is currently unknown why Dbr1p is specific while other enzymes in this superfamily are not. Easy access to substrates for Dbr1p has previously been a major roadblock in conducting biochemical investigations of the enzyme. This lab has developed a robust method for synthesizing backbone branched RNA (bbRNA) that contain a 2'-5'-phosphodiester bond. A photolabile protecting group is employed during synthesis to create a branch point residue, from which a 2'-5'-phosphodiester bond can be formed. Improvements to the photodeprotection method are discussed. With ready access to easily modifiable bbRNA, the substrate requirements of Dbr1p are investigated. A variety of non-native bbRNA are incubated with Dbr1p and the change in catalytic activity is observed. Dbr1p is able to cleave a wide variety of backbone branched substrates but at a much slower rate than a traditional 2'-5'-phosphodiester bond. Noncleavable analogues of bbRNA have been synthesized that contain a 2'-5'-triazole linkage, called<br>click branched RNA (cbRNA). These RNAs interact with Dbr1p and are shown to be competitive inhibitors. The strength of inhibition is dictated by the size of the click branched inhibitor and kinetic analysis is performed using a fluorescence assay. Dbr1p has been shown to be important in microRNA biogenesis through the mirtron pathway. Short oligomers are synthesized that are comprised of only the guide strand sequence of a miRNA. The 5'-terminus is linked to an internal 2'-OH to form a 2'-5'-phosphodiester bond that is cleavable by Dbr1p. These lariat structures are used as siRNA and delivered into cells. Repression of mRNA and protein expression in human cancer cell lines is observed. These miRNA mimics have the potential to be used as a therapeutic, similar to how siRNA is currently<br>being used. Splicing occurs upstream of Dbr1p action. Lariat introns are formed during this process through the spliceosome and RNA is at the core of the spliceosomal catalytic machinery. With easy access to bbRNA, we are able to model the spliceosome immediately after the first catalytic step, branching. We set out to develop an assay to determine if the RNA at the core of the spliceosome is catalytic in a protein-free environment. Detailed analysis of the strength of RNA duplexed to bbRNA is performed using melting temperatures. As the spliceosomal RNA is not<br>entirely duplexed, we created constructs that mimic these interactions. The melting temperatures are determined for bbRNA duplexes to see how the bbRNA structure might stabilize or destabilize a duplex interaction. Binding and cleavage assays are performed to see if spliceosomal RNAs<br>can cause catalysis on bbRNA in a protein-free environment. This thesis focuses on understanding the processing of lariat introns upstream (splicing) and downstream (Dbr1p<br>cleavage) of intron formation. <br>