Molecular basis of the DExH-box RNA helicase RNA helicase A (RHA/DHX9) in eukaryotic protein synthesis.

摘要

RNA helicase A (RHA/DHX9) is a cellular protein and member of the DExH/D-box RNA helicase family that is necessary for the translation of pathogenic retroviral transcripts and the proto-oncogene junD. The known characteristic of RHA-mediated protein synthesis is the select recognition and association of RHA with the distinguishing 5' termini of retroviral and junD transcripts. These mRNAs harbor the distinct 5' RNA motif known as the posttranscriptional control element (PCE). The PCE functions in cis to stimulate translation activity via the canonical cap-dependent scanning mechanism that defines eukaryotic protein synthesis. The main gap in knowledge is how RHA, as the host effector of the PCE, engages a RNP complex that facilitates translation activity. In this study, RHA was identified to engage a unique RNP that confers a novel role for its activity in cap-dependent translation during cell stress. Here, RHA was demonstrated to selectively interact with the non-canonical CBP80/20 cap-binding protein. Notably, this interaction was maintained during serum deprivation, torin 1-mediated mTOR inhibition and HIV-1 expression, all mechanisms that evoke cell stress and suppression of the canonical eIF4E cap-dependent translation. This effect correlated with sustained interactions between RHA, CBP80/20, and the target PCE transcript HIV-1 gag on polysomes. The outcome was maintained cap-dependent retroviral PCE translation. This dissertation also identified a novel function of RHA as a post-initiation effector of protein synthesis. DExH/D-box RNA helicases mediate initial translation events of 5' ribosome binding and scanning. A reverse genetics approach showed that the N-terminal double-stranded RNA binding domains of RHA are critical for these canonical family activities. Notably, there is a role for the C-terminal arginine-glycine-rich domain of RHA in 80S ribosome stabilization. This post-initiation translation activity is fundamental to the engagement of RHA with polyribosomes and completion of the translation process. A third major finding of this dissertation was the identification of RHA to engage multiple RNP states that regulate its translation activity. Here, RHA self-associates, a homopolymeric-binding event that impaired translation cofactor binding. This result indicated a role for RHA self-association in the regulation of its translation RNP formation. In addition, a select interaction between RHA and related DExH-box RNA helicase DHX30 was identified. This heteropolymeric-binding event was distinct in that it characterized a RHA RNP engaged with polyribosomes. This result indicated a role for a RHA-DHX30 association in the direct regulation of RHA translation during active protein synthesis. Collectively, the data obtained here elucidate the molecular basis of RHA function in protein synthesis and suggest several new paradigms for the eukaryotic translation process. These include: a molecular basis for maintained cap-dependent translation during cell stress, the significance of DExH/D-box RNA helicases in post-initiation translation control, and a role for distinct homo- and hetero-polymeric binding events in the regulation of RNP dynamics in translation. Future studies will connect the molecular findings of this research with the clinical significance of RHA. Our objective is to provide an understanding of the relationship between RHA, eukaryotic protein synthesis and cell biology that informs therapeutic approaches for animal and human health.