Changes in polysome loading as a consequence of RHA downregulation
ABSTRACT: RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5’ untranslated region (UTR). This interaction represents an “RNA switch” that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays. Changes in cytoplasmic RNA steady state abundance was monitored as well. Sixty nine genes exhibit decreased transcript polysome association when subjected to RHA downregulation. A majority of the transcripts that experienced a reduction in the polysome fraction had no significant change in their cytoplasmic abundance (45 genes). Keywords: gene expression array-based Overall design: Cytoplasmic lysates of cells treated with RHA targeted or non-silencing control siRNAs were separated by sucrose density gradient centrifugation. Ribosomal RNA profiles were generated, fractions containing polysomes were collected, and RNA was extracted.