IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

While multiple cellular factors such as competing cap-binding proteins complicate analysis in cells, our in vitro mRNA binding assays reveal a clear cap0-mRNA binding advantage conferred by IFIT1 heterooligomerisation. When examined in our reverse transcriptase inhibition assays apparent IFIT1 binding was much less efficient for the structured cap0-ZV reporter than for the less structured globin mRNA, and in fact on the ZV construct failed to reach saturation (Figure 4 ). This is not due to incomplete capping of the mRNA as saturation is possible when IFIT3 is present. Instead, it is more likely that the reverse transcriptase may remove a proportion of the bound IFIT1 as it proceeds in a 5 to 3 direction resulting in the production of a full-length signal even if IFIT1 was initially bound. We conclude therefore that IFIT3, and the IFIT2:IFIT3 heterodimer, stabilise the interaction of IFIT1 with the cap0 mRNA in such a way that it is no longer removed by the reverse transcriptase. Interestingly, there was no evidence of a shift in the IFIT dependent toeprint to indicate that the other IFITs in the complex were interacting with the mRNA downstream of the IFIT1 cap0 binding cleft. IFIT5 changes conformation when transitioning between the apo-and RNA-bound state (8), positioning key residues for optimal RNA binding. The crystal structures of IFIT1 bound to different short RNAs show the protein is in a similar closed conformation as the RNA-bound structure of IFIT5 (9) . It is therefore likely that IFIT1 cycles through a similar open/closed conformation to interact with RNA. Binding of IFIT3 may promote the closed conformation of IFIT1, stabilising its interaction with target RNAs. Future efforts will focus on obtaining high-resolution structures of IFIT1:IFIT3 and IFIT1:IFIT2:IFIT3 complexes with and without RNA to address these questions.