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

Abstract

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.innate immunity | poly-U tailing | protein-RNA interaction | RNA post-transcriptional modifications | tRNA processing I nnate immune responses provide a front-line defense against pathogens. Unlike adaptive immune responses, innate immunity relies on general principles of discrimination between self and pathogen epitopes to trigger pathogen suppression (1). Pathogen-specific features that can provide this discrimination come under evolutionary selection to evade host detection, and in turn, host genes adapt new recognition specificities for pathogen signatures. Among the most clearly established targets of innate immune response recognition are nucleic acid structures not typical of the host cell, such as cytoplasmic double-stranded RNA (2). Detection of a pathogen nucleic acid signature robustly induces type I IFN, which activates a cascade of pathways for producing antiviral effectors (3).Cytoplasmic viral RNA synthesis occurs without cotranscriptional coupling to the 5′-capping machinery, which acts pervasively on host cell nuclear RNA polymerase II transcripts (4, 5). Eukaryotic mRNA 5′ ends are first modified by addition of a cap0 structure containing N7-methylated guanosine, which is joined to the first nucleotide (nt) of RNA by a 5′-5′ triphosphate linkage (7mGpppN). In higher eukaryotes including humans, cap0 is further modified by ribose 2′-O-methylation of at least 1 nt (7mGpppNm, cap1) and sometimes 2 nt (7mGpppNmpNm, cap2). Cap0 addition makes essential contributions to mRNA biogenesis and function in steps of mRNA splicing, translation, and protection from decay (4, 5). In contrast, the biological role of mRNA cap0 modification to cap1 and cap2 structures is largely enigmatic. Some viruses encode enzymes for 7mGpppN formation and, less frequently, the ribose 2′-O-methylation necessary to generate cap1 (6). Recent studies show that virally encoded cap 2′-O-methyltransferase activity can inhibit the innate immune response (7-11).The IFN-induced protein with tetratricop...

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

“…1B) to include RNAs with post-transcriptionally added nucleotides, such as the 3 ′ CCA of tRNAs or poly(U) tails (Malecki et al 2013;Katibah et al 2014). Like other RTs and DNA polymerases, TGIRTs can add a small number of extra nontemplated nucleotides to the 3 ′ ends of cDNAs (referred to as terminal transferase activity) (Clark 1988;Golinelli and Hughes 2002).…”

“…7B). In contrast to retroviral RTs, which terminate at base modifications that affect Watson-Crick base-pairing interactions (Burnett and McHenry 1997;Ansmant et al 2001;Jackman et al 2003), TGIRTs frequently read through a number of such modifications (e.g., m 1 A58 and m 1 G9) by misincorporation, with the spectrum of misincorporated nucleotides characteristic of the modification (Katibah et al 2014;Shen et al 2015). tRNA-protein complexes have been identified previously in human sera as autoantigens in patients with autoimmune diseases, a wellstudied example being HisGUG, which is bound to histidyl-tRNA synthetase in the polymyositis-specific autoantigen Jo-1 (Hardin et al 1982;Mathews and Bernstein 1983;Rosa et al 1983).…”

“…We showed that TGIRT-seq enables miRNA profiling with less bias than two commercial RNAseq kits that rely on RNA ligation for RNA-seq adapter addition (Mohr et al 2013). Recently, we used TGIRT-seq to profile RNAs associated with the human interferon-induced protein IFIT5 (Katibah et al 2014); to identify tRNAs associated with a yeast ribosome quality control complex (Shen et al 2015); and in combination with a demethylase, for quantitative profiling of cellular tRNAs (Zheng et al 2015). These studies demonstrated that TGIRT-seq yields full-length reads of highly structured RNAs, including precursor and mature tRNAs, enables the quantification of charged and uncharged tRNAs, and facilitates the analysis of post-transcriptional modifications, including base modifications on the Watson-Crick face, many of which can be read through and result in distinctive patterns of misincorporated nucleotides.…”

“…In the initial TGIRT-seq method, the cDNAs with an RNA-seq adapter linked by TGIRT during reverse transcription were size-selected on a denaturing polyacrylamide gel and circularized with CircLigaseII ssDNA Ligase (Epicentre) prior to PCR amplification (Mohr et al 2013;Katibah et al 2014;Shen et al 2015;Zheng et al 2015). Although this procedure remains useful for RNA-seq of specific RNA size classes or homogenously sized RNA fragments in procedures like HITS-CLIP or ribosome profiling, disadvantages include (1) size limitations introduced by CircLigase, whose efficiency decreases for longer cDNAs (Epicentre product literature); (2) a gel-purification step, which is time consuming and results in loss of material; and (3) the use of hazardous chemicals, such as phenol and chloroform.…”

“…In the previous TGIRT-seq method, referred to as the small RNA/CircLigase method, cDNAs were linked by TGIRT template-switching to an RNA-seq adapter containing the complements to both the Illumina Read 2 (R2R) and Read 1 (R1R) primer-binding sites, gel purified and then circularized with CircLigase prior to PCR amplification (Katibah et al 2014;Shen et al 2015;Zheng et al 2015). In contrast, in the new TGIRT-seq method developed here, the cDNAs with an attached R2R adapter sequence are processed into RNA-seq libraries without size selection by ligating a 5 ′ -adenylated (5 ′ App) DNA oligonucleotide containing the R1R adapter to the cDNA 3 ′ end with Thermostable 5 ′ AppDNA/RNA Ligase (New England Biolabs).…”