The effect of post-transcriptional modifications in U2 and U6 on RNA X formation. (A) Authentic human U6 snRNA is active in RNA X formation. (IV U6) U6 made by in vitro transcription; (NE U6) U6 purified from nuclear extracts. Lanes marked NE U6 + dA Br or IV U6 + dA Br display RNAs from reactions that received a Br containing deoxy substitutions at A21 and G20. The position of RNA X formed with IV U6 is indicated to the right. The position of RNA X formed with NE U6 is marked by an arrow. The size difference between the two RNA X molecules is attributable to the larger size of NE U6 compared with IV U6 (seven nucleotides). (B) A conserved pseudouridine in U2 enhances formation of RNA X. (U2) U2 snRNA without any modifications was used in RNA X formation; (Ψ) U2 snRNA containing a pseudouridine at position 34. The positions of RNA X and U6 are indicated to the right. Reaction temperatures are indicated above each set of reactions.
