Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA.

Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association. The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus. Methylation was consistent with the current secondary structure model. Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive. Thus, most telomerase is not base paired to telomeres in vivo. Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions. The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence. For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern. Thus, as expected, comparative analysis gives the structure of the RNA in vivo.