Talk abstracts

Talk on Friday 03:15-03:30pm submitted by Deepak Patil

Identification of re-capping substrates for the cytoplasmic Capping enzyme complex

Chandrama Mukherjee (Molecular and Cellular Biochemistry, The Ohio State University), Deepak Patil (Molecular and Cellular Biochemistry, The Ohio State University), Brian Kennedy (Molecular and Cellular Biochemistry, The Ohio State University), Baskar Bakthavachalu (Molecular and Cellular Biochemistry, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University), Daniel R. Schoenberg (Molecular and Cellular Biochemistry, The Ohio State University)

Abstract:
Massively parallel sequencing has identified a large number of capped small RNAs and 5’ truncated forms of RefSeq transcripts. We previously described a cytoplasmic capping enzyme complex that contains capping enzyme and a kinase that converts 5’-monophosphate ends to a diphosphate capping substrate. Targets for cytoplasmic capping were identified by first overexpressing a tet-inducible dominant-negative form capping enzyme that is restricted to the cytoplasm (DN-cCE). Cytoplasmic RNA from control and induced cells was then treated with Xrn1 to degrade uncapped mRNAs and analyzed on Affymetrix Human Exon 1.0 arrays. Scoring was based on changes in probe intensity as a function of position on each RefSeq gene to derive a factor (alpha) that could be compared between sets. Transcriptome-wide changes were only evident in Xrn1-treated samples, indicating the general impact of DN-cCE is limited. This analysis identified 2,666 mRNAs with a population that is uncapped at any given time, 672 mRNAs that are enriched in the uncapped pool only in cells expressing DN-cCE, and 835 mRNAs common to both populations. Ten re-capping targets and 5 controls were selected for detailed analysis of changes in cap status by their; a) susceptibility to in vitro degradation by Xrn1, b) recovery following ligation of an RNA oligonucleotide onto uncapped 5’ ends and recovery with a biotin-tagged antisense primer, and c) separation on a high affinity cap-binding matrix containing immobilized heterodimer of eIF4E+eIF4G. In all cases this confirmed the cap status of re-capping targets identified on exon arrays. Re-capping substrates are enriched for products involved in general cellular metabolism and for downstream Kozak consensus translation initiation sites. In keeping with a role in translation, expression of DN-cCE reduces the ability of cells to recover from stress and increases the population of non-translating mRNPs.
Supported by PHS grant GM084177 and NSF agreement No. 0931642

Keywords: Uncapped transcriptome, cytoplasmic capping