Caracterização dos complexos de IFITS humanas e seu impacto na tradução celular

Abstract

Among the most highly expressed interferon stimulated genes are members of IFIT family (interferon-induced proteins with tetratricopeptide repeats). IFIT1 is a critical effector molecule of the antiviral cell-intrinsic immunity that binds mainly cap0 RNAs, which lack 2´-O-methylation of the first and second cap-proximal nucleotides (cap0), characteristic of non-self RNAs. After binding, IFIT1 prevents their translation by competing with the cap-binding eukaryotic translation initiation factor 4F. Despite IFIT1 can bind RNAs by itself, it was demonstrated that it interacts with several proteins, including other IFITs in cells stimulated with interferon. However, the molecular details of these interactions and the impact on IFIT1 activity wasn´t shown. The human IFITcomplexes were here reconstructed in gel filtration experiments using recombinant proteins, and their stoichiometry were determined by SEC-MALS. IFIT1 strongly interacts with IFIT3 to form a stable heterotetramer. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that interacts with IFIT1 in a heterotrimer of IFIT1:IFIT2:IFIT3. The presence of IFIT3 in these complexes increases their stability as demonstrated by differential scanning fluorimetry. IFIT3 and IFIT2:IFIT3 association also enhanced IFIT1 binding to model cap0 RNAs in toeprinting assays and the translation inhibition of its targets in vitro. By mutational analysis, the IFIT1:IFIT3 binding interface were identified as a conserved C-terminal YxxxL motif present in both proteins. Disruption of this interface abrogates IFIT3-dependent IFIT1 enhanced activity. In cells, it was demonstrated that IFIT3, but not IFIT2, stabilizes IFIT1 expression. Finally, an in vitro pulldown system were developed to identify IFIT1:IFIT2:IFIT3 interactions with cellular self mRNAs. Bound RNAs were deep sequenced and their protein expression evaluated in the context of IFIT1 overexpression. Together this work reveals new roles for IFIT3, clarifies the critical aspects of IFIT complexes assembly and function, and brings new clues to the mechanism of IFIT1-mediated cell translation inhibition.