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

As previously reported (13), IFIT1 binds cap0--globin mRNA with very high affinity (K1/2,app= 40  1.2 nM, h = 2.8  0.2). Complexing with IFIT3 or the IFIT2:IFIT3 heterodimer reduces the K1/2,app of IFIT1 on this mRNA to 19  0.8 nM (h = 2.5  0.2) and 19  0.7 nM (h = 1.98  0.1), respectively. IFIT1 heterocomplexes also has the effect of saturating the binding on the mRNA as evident in the autoradiograms. This effect was even more pronounced when the cap0-ZV reporter was analysed. On this more structured RNA the K1/2,app of IFIT1 binding was 69  1.8 nM (h = 3.5  0.3) and only reached 60% saturation. In contrast, when complexed with IFIT3 or the IFIT2:IFIT3 heterodimer IFIT1 bound the ZV reporter with a K1/2,app of 29  1.1 nM (h = 4.1  0.6) and 58  2.3 nM (h = 3.1  0.32), respectively. Again, as is clear from the autoradiograms, addition of IFIT3 or the IFIT2:IFIT3 heterodimer led to saturation of RNA binding. In all cases, and similar to our previous findings (13), the Hill coefficient was greater than 1, indicating a degree of cooperativity in IFIT1 cap0-mRNA binding.