Darville-O’Quinn, Paige – Novel Proteomic Approach for the Identification of Novel mRNA-bound Splicing Factors (Dr. Yu Lu)

The removal of non-protein coding introns from the precursor messenger RNA (pre-mRNA) transcript, through the process known as splicing, is a necessary step in gene expression.  Alternative splicing of a single pre-mRNA can result in multiple different protein isoforms, depending on the combination of exons and introns retained, thus contributing to the proteome diversity seen in eukaryotes. RNA binding proteins (RBPs) regulate this process and dysregulation can lead to a multitude of diseases, including Acute Myeloid Leukemia (AML). The alternatively spliced AML1-ETO9a protein is highly leukemogenic, leading to the development of AML, however the splicing factors involved in the production this isoform have yet to be identified. Identifying these proteins would improve our understanding AML disease progression, however current protocols for the isolation and identification of proteins associated with mRNA transcripts are limited. With modifications, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins system has the potential to specifically enrich mRNA, as a novel method of isolating RBPs for proteomic analysis. In this project, components of the CRISPR-Cas system, modified for binding single stranded RNA, were produced for future mRNA enrichment and proteomic analysis. GAPDH mRNA-specific guide RNA (gRNA) was produced, and two deactivated Cas proteins, dCas9 and dCas13a, were both expressed in E. coli BL21 (DE3) cells. Following expression, these proteins can be made to target specific mRNA sequences based on the gRNA produced. These sequences can then be pulled down, and their associated proteins identified through mass spectrometry. If successful, this method has the potential for use in identifying targets for the prevention, detection and treatment of diseases such as cancer.