Decapping and deNADding Assay Protocol

³²P-NAD-cap labeled or ³²P-m⁷G-capped RNAs were incubated with the indicated amount of recombinant proteins in decapping buffer containing 10 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM DTT, 2 mM MgCl₂ and 2 mM MnCl₂ as previously described^{39 (link)} and incubated at 37 °C for 30 min. Reactions were stopped with 30 mM EDTA. For reactions involving Nuclease P1 treatment, reactions were first extracted with phenol followed by chloroform and 1U of Nuclease P1 (Sigma-Aldrich) was added. The reactions were incubate at 37 °C for 30 min. Decapping products were resolved by PEI-cellulose TLC plates (Sigma-Aldrich) and developed in 0.5 M LiCl or 0.45 M (NH₄)₂SO₄ in a TLC chamber at room temperature^{40 (link)}. deNADding assays in Fig. 4S were similarly carried out except, following termination of the reaction with 30mM EDTA, the RNA were resolved by 15% denaturing polyacrylamide gel electrophoresis and dried. Reaction products were visualized with a Molecular Dynamics PhosphorImager (Storm860).