Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation.
Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.
Ryner
L C
LC
Department of Biological Sciences, Columbia University, New York, New York 10027.
Takagaki
Y
Y
Manley
J L
JL
D000645:Ammonium Sulfate; D002467:Cell Nucleus / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D011062:Polynucleotide Adenylyltransferase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*
D007527:Isoenzymes; D009097:Multienzyme Complexes; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase; D000645:Ammonium Sulfate