In collaboration with Projects 1, 2, 5 and Cores A and B, Project 3 has elucidated a new translation regulatory axis by comparative studies of retroviral genomes. Our results of proteomic, genetic and biochemical analysis have identified a novel role for RNA helicase A (RHA) in both retroviral and cellular genes. We have shown that RHA specifically recognizes a unique 5'terminal post-transcriptional control element (PCE) and neutralizes structural features of the 5'untranslated region (UTR) to facilitate efficient cap-dependent translation initiation. Our results of biochemical analyses and genome-wide translation profiling have identified a subset of biologically-related genes that require RHA for their efficient translation. Many of these PCE candidates are proto-oncogenes encoding a complex 5'UTR, which require RHA/PCE interaction to promote efficient translation. Our identification of the fundamental role of RHA in cellular translational control provides a platform to understand the observation that RHA dysregulation is a tumor biomarker. Our collaborative studies have identified PCE activity in six divergent retroviruses, including HTLV-1;that interaction with RHA is necessary;and this virus-host interaction is essential for efficient HTLV- 1 translation. Our additional identification of PCE activity in cellular junD provided proof-of-concept that retroviruses have adopted a host cell mechanism to achieve efficient RNA expression. We have applied the PCE/host interaction to stimulate protein output in retroviral vectors;this innovation is applicable to a wide array of gene expression systems. Our fundamental insights implicate RHA as an integrative effector in the continuum of gene expression from transcription to translation and in coordinating viral and cellular gene expression. The outcomes of the initial funding period are inextricably linked to the common PPG goal to understand virus-host interactions and mechanisms of gene regulation. A primary focus of this highly interactive Continuation is to understand the scope and regulation of the RHA/PCE translational control axis in retroviral and host genes. Specifically, we postulate the RHA regulon is an inducible translational control mechanism of selected genes, whose dvsregulation contributes to alterations of the cellular microenvironment leading to transformation and paraneoolastic disease. Our three interrelated Aims are:
Aim 1. To characterize essential features of RHA gene expression and cytoplasmic localization during cell cycle progression;
Aim 2. To examine role of RHA translational activity in osteoclast activity and Tax tumor model;
Aim 3. To assess the essential features structure/function of junD PCE in relation to the retrovirus PCE database. Long-term objectives are application of knowledge of the RHA post-transcriptional regulon to develop vectors and small molecules to selectively modulate RHA responsive genes involved in neoplastic transformation, paraneoplastic disease and retrovirus infection.
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