Fluorescent RNA Cleavage Assay

C2 RNA cleavage was performed as described previously^{26 (link),27 (link)} with minor changes. Full-length ScLas1-Grc3 variants (0–0.8 μM) were incubated with 1 mM EDTA and 50 nM fluorescein labeled C2 RNA mimic (5′-GUCGUUUUAGGUUUUACCAACUGCGGC/36-FAM/−3′) in the absence and presence of 2 mM nucleotide (ATP). Contaminating nucleotides were removed from commercial ATP (Sigma; A2383) using previously established nucleotide purification protocols^{60 ,61} . Briefly, ATP (6 mg) was resuspended in 50 mM (NH₄)HCO₃ and bound to a MonoQ 4.6/100 PE (GE Healthcare) column equilibrated with 50 mM (NH₄)HCO₃. ATP was eluted using a 50–500 mM (NH₄)HCO₃ linear gradient over 45 mL. ATP fractions were lyophilized and resuspended to 10 mM using water. Unless specified otherwise, reactions were incubated for 1 hour at 37°C and quenched with urea-loading dye. Samples were resolved on 15% polyacrylamide (8 M urea) gels in 1x tris-borate-EDTA buffer and visualized using a Typhoon FLA 9500. Representative gels are shown from three independent replicates.