RNA in vitro transcription and labeling

RNA substrates for binding studies were generated by T7 run-off transcription using the Milligan transcription method from annealed template DNA oligonucleotides (Milligan et al., 1987). The U1 stem-loop 4 substrate comprises human U1 snRNA nucleotides 137-164 followed by 34 downstream nucleotides, including the 3′-box sequence. The control RNA is a scrambled sequence of the same size. Transcription products were gel purified, enzymatically capped with the vaccinia capping enzyme (NEB) following manufacturer recommendations and labeled with fluorescein at the 3′ end. Briefly, the 3′ vicinal diol was oxidized by re-suspending the RNA in 100 μL of freshly made oxidation solution (0.1 M sodium periodate and 0.1 M sodium acetate (pH 5.0)) and incubated at room temperature for 1.5 h in the dark. The reaction was quenched by the addition of 10 μL of 3 M KCl, placed on ice for 10 min, and the resulting insoluble KIO₄ was pelleted by a brief centrifugation. A thiosemicarbazide derivative of fluorescein (100 mM in DMSO) was added to a final concentration of 50 mM and incubated at room temperature for 4 h, as previously described (Hardin et al., 2015). Labeled products were purified using a denaturing PAGE.