Talk abstracts

Talk on Friday 01:30-01:50pm submitted by Keyur Vyas

Genome wide screening for target mRNAs silenced translationally upon IFN-gamma treatment

Keyur Vyas (Center for Gene Regulation in Health and Disease, BGES, Cleveland State Univ., OH), Sujan Chaudhuri, Anton A. Komar, (Center for Gene Regulation in Health and Disease, BGES, Cleveland State Univ., OH), Douglas Leaman (Department of Biological Sciences, University of Toledo, OH), Alla Musiyenko, Sailen Barik (Department of Biochemistry and Molecular Biology, University of South Alabama, AL), Barsanjit Mazumder (Center for Gene Regulation in Health and Disease, BGES, Cleveland State Univ., OH)

Abstract:
The ribosomal protein L13a inhibits the translation of IFNã-induced ceruloplasmin (CP). Upon release from the 60S ribosome, L13a forms a GAIT (IFN-gamma activated inhibitor of translation) complex, which binds to the 29 nucleotide sequence, refered to as GAIT element, on the 3’UTR of CP mRNA for silencing. Thus following initial upregulation of CP there is a shut-off of CP after 16 hrs. We hypothesize that the translational silencing mechanism is not exclusive for CP but applies to multitude of proteins. Therefore, we conducted a genome-wide analysis using Affymetrix Genechip and Inflammation Responsive Genearray to identify other proteins. Following 0, 4, and 18 hr. IFNã treatment, polysome bound mRNA (translation active pool) and unbound mRNA (inactive pool) were isolated using sucrose gradient and processed. We focused on mRNAs that had a similar polysomal profile as CP, i.e. translationally active at 4 hr., while inactive at 18 hr. A cluster of mRNAs encoding different chemokines and chemokine receptors and molecules important in cytokine signaling was identified as common hits in the two independent approaches. The selected mRNA targets were validated using real-time PCR. Further, in silico-determined GAIT element in the 3’UTR of the selected mRNAs were confirmed as functional cis-acting elements for translational silencing by luciferase reporter assay. The newly identified target mRNAs also required L13a for silencing. Thus this cohort of mRNAs may represent a new inflammation responsive post-transcriptional operon regulated directly at the level of translation in order to control inflammation.

Keywords: translation control, microarray, IFN gamma