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

We and others have previously demonstrated that IFIT1 binds preferentially to mRNA with a cap0 structure at the 5 end (13, 14) . Using our purified complexes, we next examined if the interaction of IFIT1 with cap0 mRNA was altered when part of a larger IFIT1:IFIT3 or IFIT1:IFIT2:IFIT3 complex. We used a primer extension inhibition assay to monitor the IFIT1/cap0 mRNA interaction as previously described (13) . An advantage of this technique over other methods to analyse protein-RNA interactions, such as electrophoretic mobility shift assays, is that the primer extension reaction is performed in equilibrium binding conditions. IFIT1 alone or as part of an IFIT heterocomplex was incubated with the in vitro transcribed and capped model -globin and ZV mRNAs prior to the addition of a radiolabelled primer that binds within the Fluc mRNA sequence. A reverse transcription reaction is performed in which a full-length cDNA is produced in the absence of IFIT1, whereas a 7 nucleotide truncated cDNA, corresponding to the length of the IFIT1 RNA-binding surface, is produced in the presence of IFIT1 (13) . The cDNA products are subsequently separated by denaturing PAGE and detected by autoradiography. IFIT protein complexes shown in Figure 3B were used in the binding reactions. Representative autoradiographs are shown in Figure 4A . Quantification of the cDNA products was performed as described in the Materials and Methods and the binding curves shown in Figure 4B and C show the fraction of RNA bound at varying IFIT concentrations.