Splicing and transcription are crucial biological processes involved in development, contributing to the generation of diverse transcripts and proteins. The resulting protein diversity gives rise to function- and sex-specific differences among cells, which play significant roles in disease response and pharmacological outcomes. It is not well understood how splicing and transcription is co-regulated by transcription factors (TFs) and RNA-binding proteins (RBPs). In particular, it is not known how co-transcriptional splicing occurs at the correct genomic locations. We hypothesize that specific TFs bind to specific DNA and RNA sequences to regulate coordinated co-transcriptional splicing at specific chromatin sites. As such, we investigated the role of various protein domains of the transcription factor CLAMP (Chromatin-linked adapter for MSL proteins) in DNA and RNA binding. CLAMP is an ideal candidate for gaining insight into the role of TF protein-DNA and -RNA interactions since it directly binds to RNA and DNA. Earlier studies in CRISPR-generated fly mutants have also shown that deletion of the N-terminal Zinc Finger domain and Prion-like domain (PrLD) results in CLAMP-dependent splicing changes. Furthermore, deletion of the PrLD domain results in early embryonic lethality. However, presence of the initial 6Qs (glutamines) in the PrLD domain rescues lethality and allows flies to survive until adulthood. In the lab, we had already expressed PrLD-deleted CLAMP proteins in the bacterial expression system with the help of Fawzi Lab. We also used a PCR-mutagenesis to generate plasmids with deletions in the Zinc Finger (ZnF) and Prion-like Domain with the initial 6Qs (PrLD+6Q) domains of the full-length and 1-300 amino acid long CLAMP. Mutant proteins using these plasmids were expressed, which were used to perform RNA and DNA gel shift assays to determine protein-nucleic acid interactions. We found that the PrLD domain of CLAMP is essential for binding to specific RNA sequences. We also identified specific RNA sequences that aid in binding to CLAMP proteins. Furthermore, full-length CLAMP has better binding efficiency than 1-300 length CLAMP.
Chen, Victoria J.,
"Transcription Factor CLAMP Regulates Splicing through Protein-Nucleic Acid Interactions"
(2023).
Summer Research Symposium.
Brown Digital Repository. Brown University Library.
https://doi.org/10.26300/9j7t-rs17
Each year, Brown University showcases the research of its undergraduates at the Summer Research Symposium. More than half of the student-researchers are UTRA recipients, while others receive funding from a variety of Brown-administered and national programs and fellowships and go …
