Purification of human U6 small nuclear RNA capping enzyme. Evidence for a common capping enzyme for gamma-monomethyl-capped small RNAs.
To understand the mechanism of gamma-monomethyl (meppp) cap formation, we attempted to identify and purify the U6 small nuclear RNA capping enzyme. Although more than one protein was cross-linked to U6, 7SK, B2, or plant U3 RNA, only one protein of approximately 130 kDa was common to all four meppp-capped RNAs; 5 S RNA, which is an uncapped RNA, was not cross-linked to this protein. In addition to specific cross-linking with meppp-capped RNAs, an approximately 130-kDa protein was also cross-linked to 3H-labeled AdoMet. We purified the capping enzyme from a HeLa cell S-100 extract by several successive chromatographic steps, and an approximately 130-kDa protein was purified along with the capping activity. The capping activity and the approximately 130-kDa protein also cosedimented on a glycerol gradient. The purified enzyme catalyzed meppp cap formation of U6, 7SK, B2, and plant U3 RNA, and this enzyme is probably responsible for the capping of multiple RNAs in vivo. The capping activity is distinct from U6 snRNA N6-adenosine methyltransferase, and this is the first methyltransferase to be purified that methylates gamma-phosphate residues in RNAs.
Shimba
S
S
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Reddy
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R
D002384:Catalysis; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D012315:RNA Caps; D012342:RNA, Small Nuclear; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C087091:U6 small nuclear RNA methyltransferase; C022556:mRNA (2'-O-methyladenosine-N6-)-methyltransferase