Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5

Significance This study defines the biochemical and biological specificity of RNA 5′-end discrimination by the human innate immune response protein denoted IFN-induced protein with tetratricopeptide repeats 5 (IFIT5). Cellular and recombinant protein interaction assays revealed an adaptable RNA binding site that, considering both WT and mutant proteins, can broadly accommodate and distinguish phosphate-containing 5′ ends. Comprehensive profiling of IFIT5-associated cellular RNAs by thermostable group II intron reverse transcriptase sequencing identified a broad spectrum of IFIT5-bound precursor and processed tRNA transcripts and revealed a surprisingly flexible order of human tRNA processing reactions. Our findings for IFIT5 illuminate new structural specificities of protein–RNA recognition important for innate immunity. Interferon (IFN) responses play key roles in cellular defense against pathogens. Highly expressed IFN-induced proteins with tetratricopeptide repeats (IFITs) are proposed to function as RNA binding proteins, but the RNA binding and discrimination specificities of IFIT proteins remain unclear. Here we show that human IFIT5 has comparable affinity for RNAs with diverse phosphate-containing 5′-ends, excluding the higher eukaryotic mRNA cap. Systematic mutagenesis revealed that sequence substitutions in IFIT5 can alternatively expand or introduce bias in protein binding to RNAs with 5′ monophosphate, triphosphate, cap0 (triphosphate-bridged N7-methylguanosine), or cap1 (cap0 with RNA 2′-O-methylation). We defined the breadth of cellular ligands for IFIT5 by using a thermostable group II intron reverse transcriptase for RNA sequencing. We show that IFIT5 binds precursor and processed tRNAs, as well as other RNA polymerase III transcripts. Our findings establish the RNA recognition specificity of the human innate immune response protein IFIT5.