Abstract 3159: RPS6 controls the translation of 5’ terminal oligopyrimidine tract-containing mRNAs through a physical interaction that regulates the loading of these messages on the polysome

Abstract

Abstract The prevailing paradigm for mTOR signaling implicates RPS6 activation in translational control of mRNAs characterized by the presence of an oligopyrimidine tract (5’ terminal oligopyrimidine) within the 5’ untranslated region. These messages containing an oligopyrimidine tract are important for ribosomal biogenesis as they encode most of the translational apparatus. However, mechanistic details of how RPS6 is involved in the regulation of these oligopyrimidine tract-containing messages are still poorly understood. We demonstrated that through small inhibitory RNA (siRNA) knockdown of RPS6, 5’ TOP mRNA were selectively upregulated in heavy polysomal fractions. The ability of RPS6 to interact with endogenous mRNA containing 5’ TOP sequences was tested by immunoprecipitation (IP) analysis using lysates from Diffuse Large B-cell Lymphoma (DLBCL) cell lines (Farage and OCI-LY10) and a specific RPS6 antibody. The association of RPS6 with 5’ TOP mRNA was interrogated using RT-qPCR to examine mRNAs isolated from the IP material using primer sets specific for multiple 5’ TOP mRNAs. We found that 5’ TOP containing mRNA were highly enriched in IP material obtained with anti-RPS6 antibody compared with the background level of amplification seen in control immunoglobulin G (IgG) IP. We constructed a heterologous green fluorescent protein (GFP) mRNA containing the 5’ TOP sequence from the RPS6 gene in the 5’ UTR. Using mRNP-IP assays, RPS6 was found to interact with the heterologous construct containing the 5’ TOP sequence when compared to the normal GFP mRNA control. A number of previous publications indicated that aberrant control of protein translation contributes to lymphomagenesis. RPS6 has been shown to control the formation of nascent ribosomal subunits and to participate in ribosomal biogenesis. An overabundance of RPS6 has been postulated to lead to an increase in the total number of ribosomes and change the profile of translated mRNAs, including messages with a low affinity for the translation machinery i.e., oncogenic mRNAs. To investigate the possibility that RPS6 is overexpressed in lymphoma, we screened a panel of DLBCL samples for RPS6 protein levels. Utilizing immunohistochemistry, we observed that RPS6 protein levels were significantly increased in DLBCL compared to B cells found in the germinal center of matched normal reactive lymph nodes. Finally, using short hairpin RNA (shRNA) targeting RPS6 we demonstrated significantly increased apoptosis in DLBCL cell lines when compared to control shRNA. Our work sheds light on how RPS6 may regulate the translation of mRNA containing a 5’ TOP sequence through a direct protein-mRNA interaction and has potential therapeutic implications for targeting RPS6 in lymphoid malignancies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3159.