My long-term career goal is to becoming an independent investigator dissecting genetics mechanisms of psychiatric and neurodevelopmental disorders using cutting-edge genetics/genomics approaches. During the mentored training period of the proposed K99 career development award, my goal is to develop the necessary technical skills and knowledge background that will prepare me to succeed as an independent investigator. With the help from all my mentors and collaborators, I am in a great position to be able to get the necessary training and preparation for the independent phase, which include 1. Further developing new genomics technologies; 2. Learning more stem cell biology techniques for conducting experiments using human induced pluripotent cells (hIPSCs); 3. Learning large-scale data analysis, integration, and network construction. In addition, the department and Yale Medical School also provide me a rich variety of courses, seminars and workshops to help me to prepare for future career, including workshops in teaching skills, scientific writing and grant application skills. My recently finished large pooled shRNA librariesscreening studies have revealed a group of genes which are important for forming neuronal progenitors cells (NPCs) from human embryonic stem cells (hESCs) and have been implicated in various neurological abnormalities. Most of these genes encode proteins with both prion-like domains (PRD) and RNA-binding domains (RRM) and play a role in RNA granule formation. To further study this group of genes and their functions in neural development, I will: 1. Determine the global effects of knocking down two genes (TAR DNA binding protein (TARDBP) and TIA1 cytotoxic granule-associated RNA binding protein-like 1 (TIAL1)) in hESCs during induction of neural differentiation, including the effects on mRNA levels, splicing patterns, and translation rates.(mentored phase) 2. Construct double shRNA libraries to systematically map how pairwise combinations of knockdowns of target genes alter early steps of neural development in vitro. By comparing phenotypes of double knock downs with those of the singles, one can generate genetic interaction (GI) map to elucidate epistatic relationships between components and define functional pathways. (mentored phase). 3. The following studies will be mainly conducted during the independent phase of the award (year 3 and onward). 3a. Perform confirmation and functional studies following double shRNA libraries screening studies. 3b.To understand the functions of PRD domains in neuronal degenerative associated genes such as TARDBP, I will construct a library of genes expressing TARDBP with a variety of single and multiple amino acid mutations in the PRD domains, including all 40 diseases associated mutations and test the ability of the various mutant genes to inhibit or facilitate NPC differentiation. In subsequent yeas selected mutant proteins will be used to as a probe to screen for gene modifiers that can rescue the deficit of NPC differentiation caused by a particular mutation of TARDBP.
I propose to use newly developed genetics/genomics technologies to systematically evaluate the functions of a group of genes coding for proteins with prion-like domains and their roles in neural development. This offers new understanding of how the mutations on those genes contribute to certain psychiatric and neurological disorders.