Poster abstracts
Poster number 115 submitted by Deepak Sharma
Biochemical characterization of the human DEAD-box RNA helicase DDX3X
Deepak Sharma (Center for RNA Molecular Biology, Case Western Reserve University), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract:
The human DEAD-box RNA helicase DDX3X is expressed in multiple tissues and has been implicated in transcription regulation, RNA export, translation, and several signal transduction pathways. Mutations and altered expression of DDX3X have been linked to the development of multiple tumors, and DDX3X is directly targeted by several different viruses. Previous studies have qualitatively shown helicase and ATPase activities for DDX3X, but many important biochemical features of this protein are unknown.
Here, we systematically characterize enzymatic activities of DDX3X. We show that DDX3X unwinds RNA-RNA, and RNA-DNA, but not DNA-DNA duplexes. DDX3X shows a significant preference for unwinding RNA-RNA substrates with 3’ unpaired regions over those with 5’ unpaired regions; a notable difference to its yeast ortholog Ded1p, which does not display this pronounced preference, despite more than 95% sequence similarity between both proteins. DDX3X also shows significantly less potent strand annealing activity than Ded1p. With both DDX3X and Ded1p, unwinding rate constants for RNA-RNA duplexes decrease with duplex lengths. However, DDX3X is able to unwind significantly longer duplexes than Ded1p, while Ded1p unwinds shorter duplexes much faster than DDX3X. The ATPase activity of both DDX3X and Ded1p is stimulated by ssRNA, but not by ssDNA. However, ssDNA can bind to both proteins.
Our results show biochemical activities of DDX3X that are typical for DEAD-box RNA helicases. However, the functional differences between Ded1p and DDX3X with respect to substrate preferences, strand annealing, and unwinding of long duplexes, indicate that biochemical features can markedly vary even between highly similar DEAD-box helicase orthologs.
Keywords: DDX3X, DEAD BOX HELICASE, BIOCHEMISTRY