Cap methyltransferase selective binding and methylation of GpppG-RNA are stimulated by importin-alpha.
We screened a human cDNA library for proteins that bind mRNA cap methyltransferase (MT) and isolated nuclear transporter importin-alpha (Impalpha). This direct association was confirmed by glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and nuclear colocalization. In gel shift assays, MT selectively bound RNA containing 5'-terminal GpppG, and binding was inhibited by GpppG and not by m(7)GpppC. Impalpha markedly enhanced MT binding to GpppG-RNA and stimulated MT activity. MT/RNA/Impalpha complexes were dissociated by importin-beta, which also blocked the stimulation of cap methylation by Impalpha. The presence of RanGTP but not RanGDP prevented these effects of importin-beta. These findings indicate that importins play a novel role in mRNA biogenesis at the level of cap methylation.
Wen
Y
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Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
Shatkin
A J
AJ
D002467:Cell Nucleus / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D015226:Dinucleoside Phosphates; D028884:Karyopherins; D009687:Nuclear Proteins; D012315:RNA Caps; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; C062229:7-methyl-diguanosine triphosphate; D006160:Guanosine Triphosphate; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D020931:ran GTP-Binding Protein