A BIOCHEMICAL ANALYSIS OF THE RNA BINDING ACTIVITY OF Not4 OF THE CCR4-NOT COMPLEX IN YEAST

Open Access
Author:
Lu, Bonnie Boshen
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Joseph C. Reese, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • Not4
  • Ccr4-Not
  • yeast
  • RNA binding
  • Fluorescence polarization assay
  • Electrophoretic mobility shift assay
Abstract:
Ccr4-Not is a multi-functional, multi-protein complex consisting of nine subunits (Ccr4p, Caf1p, Caf40p, Caf130p, and Not1-5p) and plays a role in nuclear RNA polymerase II transcription and cytoplasmic mRNA degradation. Not4, one of the subunits of the complex, is an E3 ubiquitin ligase with an RNA recognition motif (RRM). The RRM is a common protein domain in eukaryotes and is used for the binding of a variety of RNA sequences and proteins. However, it has not yet been shown whether Not4 binds RNA or whether the RRM of Not4 binds RNA. In this research project, we cloned the full coding region of NOT4 and the NOT4 RRM coding region into expression vectors to over-express the proteins in E. coli. The expressed proteins were purified and used in in vitro RNA binding assays to determine RNA binding activity. The in vitro RNA binding assays used were the fluorescence polarization (FP) assay and electrophoretic mobility shift assay (EMSA). The Not4 full-length protein binding assays gave variable dissociation constants. The Not4 RRM binding assays did not show RNA binding under the conditions examined. These results show that Not4 binds RNA but does not eliminate the possibility that the RRM contributes to this function. We propose that the variable RNA binding activity seen with the Not4 full-length protein may be due to either experimental conditions that interfere with protein folding or protein–RNA binding, and that the lack of RNA binding activity seen with the Not4 RRM may be due to additional protein domain requirements for RNA binding.