Poster abstracts

Poster number 126 submitted by Frank Tedeschi

Genome-wide identification of cellular RNA targets of the DEAD-box helicase Ded1p

Frank A. Tedeschi (Department of Biochemistry and Center for RNA Molecular Biology, Case Western Reserve University), Ulf-Peter Guenther (Department of Biochemistry and Center for RNA Molecular Biology, Case Western Reserve University), Akshay Tambe (Department of Biochemistry and Center for RNA Molecular Biology, Case Western Reserve University), Eckhard Jankowsky (Department of Biochemistry and Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
DEAD-box RNA helicases use ATP to bind and remodel RNA and RNP complexes. In vitro, most DEAD-box proteins display little sequence specificity, but in the cell many of these enzymes are thought to target specific RNAs. Although most eukaryotic DEAD-box proteins have been implicated in defined cellular processes, it is often not known which RNAs are targeted and where targeted RNAs are bound. The absence of this critical in¬formation greatly complicates the under¬standing of biological functions of DEAD-box proteins on a molecular scale. Here, we define binding sites in RNA targets of the DEAD-box helicase Ded1p from Saccharomyces cere¬visae. We combined in vivo crosslinking of genomically encoded, histidine-biotin (HB) affinity tagged Ded1p with protein purification under denaturing con¬ditions, followed by cDNA library genesis from crosslinked RNAs and Next Generation Sequencing. Ded1p has been implicated in various cellular processes, including translation initiation and ribosome biogenesis. Accordingly, we find that Ded1p binds (pre-) ribosomal RNA and a large cross-section of expressed mRNAs. The number of mRNA sequence reads correlates broadly with the mRNA expression level, consistent with a role of Ded1p as a general translation initiation factor. Bindings sites of Ded1p are distributed along the entire length of mRNAs, and many are found in the ORF. The binding sites do not have an apparent sequence signature, however a major binding site of Ded1p is seen slightly downstream of the initiation codon.

Keywords: RNA, CLIP, DEAD-box