An MST-based assay reveals new binding preferences of IFIT1 for canonically and noncanonically capped RNAs.

IFITs (interferon-induced proteins with tetratricopeptide repeats) are components of the innate immune response that bind to viral and cellular RNA targets to inhibit translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5' ends. IFIT1 preferably binds RNAs modified with the m7G cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize noncanonical RNA 5' ends, including hypermethylated and noncanonical RNA caps. Further insights into the structure-function relationship for IFIT1-RNA interactions are needed but require robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis of fluorescently labeled protein as a function of increasing ligand concentration, is applicable to RNAs of various lengths and sequences without the need for their labeling or affinity tagging. Using the assay, we examined 13 canonically and noncanonically 5'-capped RNAs, revealing new binding preferences of IFIT1. The 5' terminal m6A mark in the m7G cap had a protective function against IFIT1, which was additive with the effect observed for the 2'-O position (m6Am cap-1). In contrast, an increased affinity for IFIT1 was observed for several noncanonical caps, including trimethylguanosine, unmethylated (G), and flavin-adenine dinucleotide caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge of the innate immune response mechanisms and the more effective design of chemically modified mRNAs. © 2025 Spiewla et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Citation

Tomasz Spiewla, Katarzyna Grab, Anais Depaix, Kamil Ziemkiewicz, Marcin Warminski, Jacek Jemielity, Joanna Kowalska. An MST-based assay reveals new binding preferences of IFIT1 for canonically and noncanonically capped RNAs. RNA (New York, N.Y.). 2025 Jan 22;31(2):181-192

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PMID: 39643445

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