Infections that generate capped RNA lacking 2O methylation around the first ribose are severely affected by the antiviral activity of Type I interferons. methylate their RNA at the 2O position of the cap and viruses generating uncapped RNA with 5 triphosphate groups are inhibited by an antiviral complex of different IFIT proteins. How IFIT proteins restrict viruses lacking 2O LRRK2-IN-1 methylation at the RNA cap remained unclear. We used a mass spectrometry-based approach to identify proteins binding to capped RNA with different methylation says. We found that IFIT1 directly binds to capped RNA and that this binding was dependent on the methylation state of the cap. Having recognized IFIT1 as being central for acknowledgement of 2O-unmethylated viral RNA we further examined the mode of action of IFITs and and luciferase, in 293T cells and performed affinity purifications using OH-RNA, PPP-RNA and CAP-RNA. Remarkably, only human and murine IFIT1 were detected when CAP-RNA was used as bait (Fig. 2a, b), suggesting that IFIT1 mediates binding of the IFIT complex to CAP-RNA. Consistent with the MS analysis, IFIT5 exclusively bound to PPP-RNA but not to CAP-RNA. To exclude contribution of cellular factors to the conversation between IFIT1 and CAP-RNA we used recombinant human IFIT proteins for RNA precipitations which confirmed a direct conversation of IFIT1 with capped RNA (Fig. 2c). A structure-based modelling approach using IFIT5  as template suggested that this RNA-binding cavity of IFIT1 is usually 700 ?3 larger than that of IFIT5 (Fig. S5) C implying that IFIT1 has slightly different RNA-binding properties. However, a lysine at position 151 and an arginine at position 255 of IFIT1, two residues involved with binding the terminal 5 triphosphate group on PPP-RNA by IFIT1 and IFIT5 , were also necessary for binding of IFIT1 to capped RNA (Fig. 2d), indicating a standard similar setting of binding. Body 2 Individual and mouse IFIT1 bind right to unmethylated capped RNA. To provide additional evidence that binding of IFIT1 is indeed responsible for associating the IFIT complex to CAP-RNA, we performed AP-MS experiments on wild-type (Ifit1+/+) and mutant, Ifit1-deficient (Ifit1?/?) MEFs. The overall precipitation efficiency was comparable in both cell types, as evidenced by equivalent enrichment of the RNA-binding protein Syncrip and the cap-binding protein Ncbp1 (Fig. 2e and Fig. S4b). Ifit1c was not enriched in precipitates from Ifit1?/? MEFs, which is usually consistent with the notion that this murine Ifit complex binds to CAP-RNA through Ifit1. These results show that the specific binding properties of IFIT1 are essential for recruitment of the human and LRRK2-IN-1 murine IFIT complexes to their RNA targets. IFIT1 binding depends on the methylation status of the RNA cap To identify proteins that bind LRRK2-IN-1 capped RNA in a methylation-dependent manner we used unmethylated CAP-RNA and fully methylated CAP1-RNA as baits with IFN-treated HeLa cell lysates and quantified the captured proteins by LC-MS/MS. As expected , , most cellular proteins were significantly enriched in the CAP1-RNA bound portion (Fig. 3a, Fig. S2c). The most notable exceptions were IFITs and the cellular 2O-methyltransferase FTSJD2, both of which clearly favoured CAP-RNA (Fig. 3a, Fig. S2c and Fig. 1 c, d and f, g). We confirmed the MS data by a Rabbit Polyclonal to TSPO series of RNA precipitations followed by western blotting for endogenous proteins. Proteins associating to RNA in a 5 impartial manner, such LRRK2-IN-1 as ILF3, precipitated similarly well regardless of the RNA used (Fig. 3b). Cap N7 methylation LRRK2-IN-1 increased the association of EIF4E to RNA and methylation of the 2O position did not impair precipitation efficiency. In accordance with the MS results, IFIT1 bound well to unmethylated CAP-RNA and CAP0-RNA (N7 methylated cap) but revealed reduced binding to Cover1-RNA (N7 methylated cover and 2O methylated initial ribos). Amount 3 IFIT1 binds capped RNA within a methylation state-dependent way. We next examined the efforts of individual cover methylation sites to IFIT1 binding. To this final end, we assessed binding of luciferase-tagged murine and individual IFIT1 with either Cover-, CAP1-RNA or CAP0-. The unmethylated CAP-RNA bait captured more murine or individual IFIT1 than either from the.