ACCURATE AND EFFICIENT N-6-ADENOSINE METHYLATION IN SPLICEOSOMAL U6 SMALL NUCLEAR-RNA BY HELA-CELL EXTRACT IN-VITRO

Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modifications including a single m(6)A (N-6-methyladenosine) at posit ion 43. This A-43 residue is critical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m(6)A in the corresponding position showing that this modification is evolutionarily conserved, In this study, we show that upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to m(6)A-43. This conversion was detectable as early as 3 min after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m(6)A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m(6)A formation; however, the integrity of the 3' stem-loop was necessary for efficient m(6)A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m(6)A methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA, The enzyme that catalyzes m(6)A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts.