Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

Sneha Rath; Eliza Prangley; Jesse Donovan; Kaitlin Demarest; Yigal Meir; Ned Wingreen; Alexei Korennykh

To begin deciphering the mechanism of protein synthesis regulation 2-5AMD, we examined whether it affects polysomes in WT and RNase L -/cells by sucrose gradient sedimentation. In the presence of immunogenic dsRNA (poly-I:C), the 80S monosome peak became dominant within 30 minutes, whereas polysomes progressively and accumulation of the 80S monosomes during 2-5AMD is a signature of inhibited initiation of capped mRNAs. A similar polysome change takes place upon deletion of the RNA helicase DHX29 involved in recognition of the 5'-untranslated region (5'-UTR) (Parsyan et al., 2009) or inhibition of the mammalian target of rapamycin (mTOR) that maintains translation initiation active (Gandin et al., 2014) . The 80S species that form upon DHX29 and mTOR defects are non-translating, as are the 80S species formed during 2-5AMD ( Fig. 1C; Fig. S1A ). Non-translating 80S species devoid of mRNA form readily in A549 cells following translation release with puromycin ( Inhibition of mTOR kinase is a common strategy to arrest bulk translation during stress responses (Hsieh et al., 2012; Zoncu et al., 2011) , suggesting that inhibition of translation initiation by 2-5AMD could depend on mTOR. To test this link, we assessed mTOR activity by measuring phosphorylation of the translation initiation factor 4E-BP1 whose phosphorylation by mTOR is required for translation initiation (Feldman et al., 2009 ). Activation of 2-5AMD did not affect 4E-BP1 phosphorylation, whereas a control treatment with the small molecule mTOR inhibitor INK128 (Feldman et al., 2009) worked (Fig. 2B ). Considering that 2-5A and INK128 inhibited bulk translation comparably but with different effects on 4E-BP1 phosphorylation ( Fig. 2A-B) , our data suggest that 2-5AMD inhibits translation initiation independently from mTOR.