Poster abstracts

Poster number 9 submitted by Deepak Patil

Identifying the 5’ ends of recapping targets by deep sequencing

Deepak P Patil (Center for RNA Biology, Department of Molecular and Cellular Biology, The Ohio State University), Ralf Bundschuh (Center for RNA Biology, Department of Physics and Biochemistry, The Ohio State University), Daniel R Schoenberg (Center for RNA Biology, Department of Molecular and Cellular Biology, The Ohio State University)

Abstract:
Decapping, exonuclease trimming and endonuclease cleavage generate RNAs with a 5’ monophosphate end. In mammalian cells these products are degraded by Xrn1, they can be stored uncapped or they can undergo recapping by the cytoplasmic capping enzyme complex. Capturing sites of decapping and cleavage, collectively known as the degradome, provides us clues to the events occurring in the lifetime of the transcriptome. We have developed an efficient deep sequencing strategy to specifically amplify and sequence uncapped 5’ ends. With some modifications, this technique can also be adopted for also detecting capped 5’ ends. The method uses an RNA adapter to tag the 5’ end of monophosphorylated RNAs followed by cDNA synthesis using a 5’ monophosphorylated N6 random primer yielding 5’ monophosphorylated cDNA fragments. cDNAs with sequence complementary to the 5' RNA adapter undergo a second-strand synthesis using an RNA adapter specific DNA oligo and a thermophilic DNA polymerase. The double-stranded cDNAs are 3' adenylated and ligated to adapters followed by a low cycle PCR amplification for deep sequencing. Phosphorylation of the cDNA strand selectively allows for ligation of the DNA adapter. The PCR products (150-400 bp) are gel eluted and subjected to qPCR analysis to verify the specific amplification of 5' ends of the known uncapped RNA molecules such as MAPK1, EXOSC1, 28S and 18S rRNAs. The method is highly quantitative, sensitive and precise in capturing known amounts of spiked 5’ monophosphorylated luciferase RNA down to attograms levels with a remarkably low intra-sample variation (Coefficient of variation=0.07, <1 is considered low variance). With barcoding, this method can be easily adopted for multiplexing several samples and is currently being used to capture the RNA recapping sites in mammalian cells.

Supported by grant R01 GM084177 from the National Institute of General Medical Sciences of the National Institutes of Health.

References:
Mukherjee C, Patil DP, Kennedy BA, Bakthavachalu B, Bundschuh R, Schoenberg
DR. Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability. Cell Rep. 2012 Aug 21. [Epub ahead of print] PubMed PMID: 22921400.

Affymetrix ENCODE Transcriptome Project; Cold Spring Harbor Laboratory ENCODE Transcriptome Project. Post-transcriptional processing generates a diversity of 5'-modified long and short RNAs. Nature. 2009 Feb 19;457(7232):1028-32. Epub 2009 Jan 25. PubMed PMID: 19169241; PubMed Central PMCID: PMC2719882.

Karginov FV, Cheloufi S, Chong MM, Stark A, Smith AD, Hannon GJ. Diverse
endonucleolytic cleavage sites in the mammalian transcriptome depend upon
microRNAs, Drosha, and additional nucleases. Mol Cell. 2010 Jun 25;38(6):781-8.
PubMed PMID: 20620951; PubMed Central PMCID: PMC2914474.

Keywords: Uncapped RNA, deep sequencing, Recapping