Investigation of splicing changes in dNab2 knockdown Drosophila neurons

Name: Katherine Shelmidine
Major: Biochemistry and Molecular Biology
Minor: Statistical and Data Sciences
Advisors: Seth Kelly, James West

Gene expression is the process by which information in genes produce functional proteins. Transcription is a critical step during gene expression where the strand of DNA for a gene is transcribed into pre-mRNA, which then undergoes splicing to remove non-coding introns. RNA binding proteins (RBPs) contribute to proper processing of RNA transcripts. Mutations in the genes encoding RBPs can lead to incorrect splicing and intron retention. This can impact the proteins being produced and lead to improper development of the central nervous system. The human ZC3H14 gene encodes an RBP which is important for neural development. Mutations in the ZC3H14 gene is linked to intellectual disability. The Drosophila RBP dNab2 is the functional ortholog of ZC3H14. Previous work showed that dNab2 loss in larval brain tissue leads to first intron retention in certain genes involved in CNS development. It is unknown which cell types experience splicing defects, and if splicing patterns vary among stages of development.

This study used a gene knockdown approach to decrease dNab2 levels, allowing flies to reach the adult stage as dNab2 null is a lethal phenotype. We also utilized the Gal4/UAS system to target neurons, testing if this specific cell type experiences altered splicing. We examined splicing in adult flies and larvae using Semi-Quantitative RT-PCR and performed qRT-PCR to determine the approximate percentages that dNab2 was knocked down. The knockdown group demonstrated different levels of unspliced transcript compared to dNab2 null. Potentially, the level of dNab2 was not lowered enough to produce the splicing defect. Alternatively, neurons might not experience altered splicing of these specific genes. Using a UAS-dicer could aid in decreasing dNab2 to the threshold needed to demonstrate altered splicing. Targeting a different cell type such as glia could also potentially yield results containing the altered splicing.

Posted in Comments Enabled, Independent Study, Symposium 2023 on April 14, 2023.