Poster abstracts

Poster number 80 submitted by Yu-Hsuan Karen Lai

Characterizing the role of the DEAD-box protein Dbp2 in RNA 3’ end processing

Yu-Hsuan Lai (Department of Biochemistry, Purdue University), Siwen Wang (Department of Biochemistry, Purdue University), Elizabeth J. Tran (Department of Biochemistry, Purdue University; Purdue University Center for Cancer Research)

Abstract:
RNA helicases functions in all aspects of RNA biology through remodeling of RNA structures and ribonucleoprotein (RNP) complexes. Among them, DEAD-box (DDX) proteins constitute the largest RNA helicase family in eukaryotes, playing critical roles at different levels of gene expression, including transcription, splicing, and RNA transport. Deregulation of many human DDX genes has been observed in various types of cancers and neurological diseases. However, the in vivo functions and precise RNA binding targets of individual DEAD-box helicases still remain largely unknown. Previously, studies from our laboratory have demonstrated that a DEAD-box protein in Saccharomyces cerevisiae, Dbp2, is an ATP-dependent RNA helicase in vitro. In vivo, Dbp2 functions in transcription termination, as loss of DBP2 results in a transcriptional read-through defect in a model gene locus(1). Our group also showed that Dbp2 facilitates assembly of single-stranded RNA-binding proteins onto polyadenylated mRNA, suggesting that Dbp2 may utilize its RNA helicase activity to promote RNP assembly and termination of nascent transcripts(2).
To determine the relationship between Dbp2 binding and transcription termination, we undertook a genome wide approach. First, we utilized individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) combined with Next-Gen sequencing (iCLIP-seq). After removal or adaptor sequences and PCR duplicates, we have more than 20 million mappable reads in all three replicates, with a mapping rate of more than 95%. This resulted in identification of 1950 mRNAs as Dbp2 binding targets. Second, we conducted RNA-seq to identify RNA 3’ end extensions in dbp2Δ cells compared to the wild type. This revealed that around 1000 genes express extended transcripts in dbp2Δ cells. Among these aberrantly expressed mRNAs, ~40% are also bound by Dbp2. The Fisher’s exact test indicates that the binding of Dbp2 and the read-through defect in dbp2Δ are significantly correlated, suggesting that Dbp2 modulate mRNA 3’ end processing through direct binding on the transcripts. This work will enable us to determine the precise function of Dbp2 in co-transcriptional processes.

References:
1. Cloutier SC et al. J Biol Chem. 2012
2. Ma WK et al. J Mol Biol. 2013

Keywords: DEAD-box helicases, transcription termination, iCLIP