Establishment of Hrb87F-TRIBE Flies and CRISPR-Cas9 Knockout S2R+ Cells as Tools to Study Hrb87F in Drosophila melanogaster

From the initial research in the laboratory, we found Hrb87F, a homology of human hnRNPA1, has potential in the regulation of circadian clock in Drosophila melanogaster and have been focusing on the study of biological pathways of Hrb87F. TRIBE, a novel technique to identify targets of RBP through base modification events of dADARcd fused with a specific RBP, was used to identify the RNA targets of Hrb87F. We succeeded in expressing Hrb87F-TRIBE proteins in S2R+ cells under the control of metallothionein promoter. Additionally, two distinct isoforms varying in length of Hrb87F were discovered and used in the research for further understanding its roles. However, a study of Hrb87F in vivo is required to fully describe its regulatory pathways in clock machinery. This study aims to prepare flies that are able to express Hrb87F TRIBE fusion protein in order to perform TRIBE analysis, which allows us to identify the targets of Hrb87F in vivo, by crossing microinjection flies carrying Hrb87F-TRIBE construct with Gal4 drivers to produce offspring expressing TRIBE proteins in certain tissues. Furthermore, we want to establish Hrb87F knockout S2R+ cell line using CRISPR/Cas9 system preparing for the study to see the alteration of cellular activities under the Hrb87F knockout.