Poster abstracts

Poster number 58 submitted by Chandrama Mukherjee

Physical separation of capped and uncapped RNA using recombinant eIF4E-eIF4G resin

Chandrama Mukherjee (Molecular and Cellular Biochemistry, Ohio State University), Deepak Patil (Molecular and Cellular Biochemistry, Ohio State University), Brian Kennedy (Molecular and Cellular Biochemistry, Ohio State University), Daniel R Schoenberg (Molecular and Cellular Biochemistry, Ohio State University)

Abstract:
The addition of 5’ cap is the first step in pre-mRNA processing and this reaction is catalyzed by the nuclear capping enzyme associated with RNA pol II. The cap plays a central role in subsequent steps of processing steps and in translational initiation, and loss of cap was thought to irreversibly commit mRNA to decay. In contrast to this notion, our lab identified a cytoplasmic population of capping enzyme can restore the cap onto RNA with a 5’-monophosphate RNAs. Using next generation sequencing several other labs have identified populations of uncapped mRNAs in the transcriptome, as well as CAGE tags mapping to internal sites of endonuclease cleavage. Most of these studies involve ligating a primer to the free 5’-monophosphate on uncapped for subsequent physical recovery or sequencing or removing the cap prior to primer ligation for mapping to CAGE tags. By definition these are indirect approaches to studying cap dynamics, and the efficiency of this process largely depends on the ligation reaction. To facilitate work in this area we are developing a facile and quantitative method of separating capped and uncapped RNAs. Most cap-recovery based strategies used to date use a monoclonal antibody that was raised to the trimethyl cap on snRNAs or one of several bacterially-expressed forms of eIF4E none of which provide quantitative separation of capped from uncapped RNA. In order to develop a rigorous and qutiantative method to separate these two populations of RNA we examined the efficacy of several different cap binding proteins. Our results show that the most effective separation of capped from uncapped RNA is achieved by combining of eIF4E plus a fragment of eIF4G. We successfully used this technique for separation of uncapped RNA from U2OS tet-on cells stably expressing K294A, a dominant negative mutant of the cytoplasmic capping enzyme.
Supported by NIH grant GM084177

Keywords: Uncap RNA, Capping enzyme, Cap bidning protein