(A) Schematic of mRNA cap structures and the proposed role of mouse IFIT1B in recognition and inhibition of mRNAs containing cap0-structures. Methylation of the first transcribed nucleotide of mRNAs, represented by an 'N', is performed by cap1-methyltransferases, such as human CMTR1 (hCMTR1). However, cap1-methyltransferase activity is absent in budding yeast. Mouse IFIT1B binds and inhibits translation of cap0-mRNAs, but its activity is blocked when mRNAs contain a cap1-structure. (B) A budding yeast growth assay for IFIT1B function. Since budding yeast lack a cap1-methyltransferase, their mRNAs are predicted to be targeted by mouse IFIT1B. Shown are 10-fold serial dilutions of yeast spotted on media in which the indicated IFIT is either not expressed (Glucose) or expressed (Galactose). Rows marked with '-' indicate an empty vector control. (C) Predicted role for hCMTR1 in blocking IFIT1B-mediated growth inhibition. (D–E) Yeast growth inhibition assays as in part B, but with galactose-induced co-expression of the indicated methyltransferase (cap1-methyltransferase (hCMTR1) wildtype or catalytic mutant (K239A)). In these panels, both IFIT and hCMTR1 expression are only induced upon grown on galactose-containing media. Protein expression data corresponding to these yeast strains grown in galactose are shown in Figure 4—figure supplement 1.