DDX3 Functions in RNA Interference through Translational Regulation

Human DDX3 is a member of the DEAD-box RNA helicases that play important roles in RNA metabolism. DDX3 and its homologs have been implicated in a variety of cellular processes, including translation. We previously found that DDX3 is recruited to cytoplasmic stress granules (SGs) under cell stress conditions, suggesting a role for DDX3 in translation. Although DDX3 is dispensable for general translation, it can facilitate translation of selected mRNAs with a long or structured 55 untranslated region (UTR). Given that the RNA helicase activity of DDX3 is required for its function in translation, we therefore propose that DDX3 may facilitate ribosome scanning by resolving secondary structures in the 55 UTR during translation initiation. Recently, it has emerged that DDX3 plays a role in the RNA interference (RNAi) pathway. DDX3 was reported to interact and colocalize with Ago protein in cytoplasmic foci, supporting a role for DDX3 in RNAi. However, the mechanism by which DDX3 affects RNAi has not been fully elucidated. We performed sucrose gradient sedimentation and cDNA microarray analysis to identify cellular mRNAs whose translation is regulated by DDX3 in HeLa cells. Among these mRNA targets of DDX3, we found that PACT is highly controlled by DDX3. PACT is a cellular dsRNA-binding protein (dsRBP), which functions as a binding partner of Dicer in the processing of microRNA precursors. We therefore assume that DDX3 may function in RNAi through translational regulation. To test this hypothesis, three specific aims are designed in this research project: Specific aim 1 will analyze the effects of DDX3 knockdown on microRNA expression profile. PACT prefers to recognize microRNA precursors and thus can facilitate microRNA processing. It is reasonable to assume that DDX3 plays a regulatory role in the processing of microRNAs. Specific aim 2 will examine whether DDX3 is required for translation of human TRBP and RDE-4 in C. elegans. TRBP is structurally related to PACT and functions as another binding partner of Dicer. TRBP mRNA contains a 488 nt-long 55 UTR, which is expected to form a stable secondary structure. Thus, it is possible that translation of TRBP is also regulated by DDX3. On the other hand, it would be interesting to see whether DDX3-mediated translational regulation of PACT is evolutionarily conserved in C. elegans. Specific aim 3 will study whether HCV core protein can perturb microRNA expression profile by targeting DDX3. MicroRNAs have been shown to be involved in the modulation of host response to HCV infection. HCV core protein interacts with DDX3 and thus can abrogate its function. It seems likely that HCV core protein may perturb microRNA expression profile by targeting DDX3. To accomplish the above-mentioned research objectives, standard procedures including lentivirus-mediated RNAi knockdown, microRNA sequencing, microRNA qRT-PCR, immunoblotting, sucrose gradient fractionation & polysome profiling, in v^-vo and in vitro translation assays, and adenovirus-mediated overexpression will be conducted. We expect to finish this proposed project within two years.

Project ID:PC10408-1511
External Project ID:MOST104-2320-B182-037