Poster abstracts

Poster number 28 submitted by Daniel del Valle-Morales

A new approach to NGS identification of capped 5’ ends

Daniel del Valle-Morales (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, USA), Guramrit Singh (Center for RNA Biology and Department of Molecular Genetics, The Ohio State University , Columbus, Ohio, USA), Ralf Bundschuh (Center for RNA Biology, Department of Physics, Department of Chemistry and Biochemistry, and Division of Hematology, The Ohio State University, Columbus, Ohio, USA), Daniel R. Schoenberg (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, USA)

Abstract:
The 5’ cap is an essential modification in mRNAs that is needed for its translation, and decapping leads to translational suppression and mRNA decay. Capping was originally thought to occur exclusively in the nucleus, however the identification of capped nonsense mediated decay intermediates of β–globin mRNA and of a cytoplasmic pool of Capping Enzyme (CE) led to the discovery of the cytoplasmic capping complex. We previously identified cytoplasmic capping targets by a combination of in vitro degradation with Xrn1 and position-dependent analysis of transcript loss using human exon arrays. These findings contributed to the hypothesis of cap homeostasis where translation of some mRNAs is controlled by cyclical decapping and recapping. Because uncapped mRNAs are susceptible to degradation and/or 5’ end trimming, it is difficult to map the exact location of recapped 5’ ends. To address this we developed a new approach based on use of the SMART reverse transcriptase and next generation sequencing to identify recapped ends. This approach takes advantage of the template switching of the SMART enzyme. The first step in the process involves ligation of a STOP oligo to uncapped mRNAs. The STOP oligo consists of an 18mer RNA oligonucleotide with 3 isomeric nucleotides (iGiCiG) at the 5’ end that prevent template switching. In the next step, SMART enzyme reverse transcribes capped mRNAs and template switches to the SMART oligo. The SMART oligo contains an eight nucleotide barcode and a 5’ adaptor. A second round of PCR incorporates a biotin tagged 5’ adaptor which is selected on streptavidin beads to enrich for 5’ ends. The last step involves incorporating the Illumina sequencing adaptors to bead recovered cDNAs with a final round of PCR. I will present results for individual steps in this process and their validation.

References:
C. Mukherjee, D. Patil, B. Kennedy, B. Bakthavachalu, R. Bundschuh, D. R. Schoenberg; Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability, Cell Reports (2012).
R. Machida, Y. Lin; Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform, PLOS one (2014).
C. Mukherjee, B. Bakthavachalu, D.R. Schoenberg; The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme, PLOS Biology, (2014).

Keywords: Cytoplasmic Capping, Sequencing