Evidence that the RNA methylation and poly(A) polymerase stimulatory activities of vaccinia virus protein VP39 do not impinge upon one another.
Vaccinia protein VP39 has two RNA modifying activities. In monomeric form, it acts as an mRNA cap-specific 2'-O-methyltransferase, specifically modifying the ribose moiety of the first transcribed nucleotide of m7G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerase, VP39 facilitates the rapid elongation of poly(A) tails that are already greater than approximately 35 nt in length. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2'-O-methyltransferase activity is not significantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleotide) RNAs were more stable (i.e., cap-independent) but were further stabilized by the presence of an m7G cap. An additional complex was observed at elevated RNA:protein molar ratios, indicating the presence of two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments indicated that RNA molecules undergoing poly(A) tail elongation by the VP55-VP39 heterodimer are not favored as cap-methylation substrates.
Gershon
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Department of Biochemistry and Biophysics, Texas A&M University, Houston, USA. pgershon@ibt03.tamu.edu
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D001665:Binding Sites; D019281:Dimerization; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D012315:RNA Caps; C088682:VP39 protein, Vaccinia virus; D014764:Viral Proteins; D012313:RNA; D008780:Methyltransferases; D011062:Polynucleotide Adenylyltransferase