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Nucleic Acids Research, 1995, Vol. 23, No. 13 2421-2426
© 1995

RNA

Accurate and efficient N-6-adenosine methylation in spliceosomal U6 small Nucelar RNA by HeLa cell extract in vitro

Shigeki Shimba, Joseph A. Bokar1, Fritz Rottman1 and Ram Reddy*

Department of Pharmacology, Baylor College of Medicine Houston, TX 77030, USA 1Department of Molecular Biology and MIcrobiology, Case Western Reserve University Cleveland, OH, USA

*To whom corespondence should be addressed

Received April 10, 1995. Accepted May 31, 1995.

Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcrlptional modifications Including a single m6A (W-6-methyladenosine) at position 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 m6A in the corresponding position showing that this modification is volutionarlly onserved. 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 m6A-43. This conversion was detectable as early as 3 mln after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m6A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m6A formation; however, the Integrity of the 3' stem-loop was necessary for efficient m6A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m6A methyrtransferase recognition and the enzyme probably recognizes secondary and or ertiary structural features in U6 snRNA. The enzyme that catalyzes m6A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyrtransferase which is also present in HeLa nuclear extracts.


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