Poster abstracts
Poster number 160 submitted by Daniel Michalski
The C-termini of Symplekin, CPSF73 and CPSF100 interact in the pre-mRNA 3' end processing core cleavage factor
Daniel Michalski (Department of Biology, University of Missouri-St. Louis), Mindy Steiniger (Department of Biology, University of Missouri-St. Louis)
Abstract:
A core cleavage complex (CCC) consisting of CPSF73, CPSF100 and Symplekin is required for 3’ end processing of all metazoan pre-mRNAs. We are characterizing in vivo molecular interactions in the CCC required for both assembly and catalysis in Drosophila. To understand which regions of the CCC components are required for complex assembly, we created stably transfected Dmel-2 cell lines expressing full-length (FL), N- and C-terminally truncated CCC factor proteins. IPs show that FL proteins are successfully incorporated into CCC containing endogenous binding partners. IP of N- and C-terminal deletion mutants reveals that the C-termini of Symplekin and CPSF73/100 are required for CCC formation. Analysis of mutant CCC activity is also being investigated. First, endogenous proteins are RNAi-depleted and mutant complexes are created with RNAi-resistant proteins. The 3’ ends of endogenous H2A mRNA from these cells are then mapped. Results indicate that the activity of Symplekin C-terminal mutant complexes correlates with their ability to form CCC. Interestingly, when only the N-terminal 271 aa of Symplekin are deleted, H2A 3’ end misprocessing is observed even though this mutant recruits CPSF73/100 more efficiently than FL Symplekin. We hypothesize that removal of the N-terminal region of Symplekin inhibits binding to the RNA pol II CTD. This reduces the local CCC concentration and causes misprocessing. We also observed partial rescue of the 3’ end processing defect when CCC include only the 200 C-terminal aa of CPSF73. This CPSF73 mutant recruits Symplekin and CPSF100 as well as FL CPSF73, but these CCC lack the CPSF73 active site. We hypothesize that including the C-terminal 200 aa of CPSF73 reduces co-depletion of endogenous Symplekin and CPSF100. Therefore, the small amount of FL CPSF73 unaffected by RNAi-depletion can form functional CCC. While these experiments are ongoing, we have already gained significant insight into CCC molecular interactions and activity.
Keywords: pre-mRNA 3 end processing, protein-protein interactions, Drosophila