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

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/261776 doi: bioRxiv preprint expressing FLAG-tagged IFIT1, FLAG-tagged IFIT5 (previously reported not to interact with other IFITs (12, 29) ), or an empty vector control. 24 hours after transfection the cells were treated with IFN-α and incubated for a further 16 hours. Preparation of nuclease treated cell lysates and pull-down experiments are described in Materials and Methods. Consistent with previous reports (38) , IFIT1 was poorly overexpressed while IFIT5 was strongly expressed ( Figure S1 ). However, as shown in Figure 1A , IFIT2 and IFIT3 co-precipitated with FLAG-tagged IFIT1, while FLAG-tagged IFIT5 did not precipitate other IFIT family members ( Figure 1B ). The full SILAC data set is available on the PRIDE server. These results independently confirm the interaction of IFIT1, 2 and 3 in IFN-stimulated cell lysates and further demonstrate that this interaction is maintained after nuclease treatment. Both IFIT2 and IFIT3 were enriched to a similar extent in the IFIT1 pull downs ( Figure 1A ).