Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase.
We have isolated cDNA clones encoding a novel RNA-binding protein that is a component of a multisubunit poly(A) polymerase from pea seedlings. The encoded protein bears a significant resemblance to polynucleotide phosphorylases (PNPases) from bacteria and chloroplasts. More significantly, this RNA-binding protein is able to degrade RNAs with the resultant production of nucleotide diphosphates, and it can add extended polyadenylate tracts to RNAs using ADP as a donor for adenylate moieties. These activities are characteristic of PNPase. Antibodies raised against the cloned protein simultaneously immunoprecipitate both poly(A) polymerase and PNPase activity. We conclude from these studies that PNPase is the RNA-binding cofactor for this poly(A) polymerase and is an integral player in the reaction catalyzed by this enzyme. The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W. (1996) EMBO J. 15, 1132-1141), raises interesting questions regarding the subcellular location of the poly(A) polymerase under study. We have reexamined this issue, and we find that this enzyme can be detected in chloroplast extracts. The involvement of PNPase in polyadenylation in vitro provides a biochemical rationale for the link between chloroplast RNA polyadenylation and RNA turnover which has been noted by others (Lisitsky, I., Klaff, P., and Schuster, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13398-13403).
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Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, University of Kentucky, Lexington, Kentucky 40546, USA.
Gupta
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JD
Hunt
A G
AG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology; D017931:DNA Primers; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011062:Polynucleotide Adenylyltransferase; D011117:Polyribonucleotide Nucleotidyltransferase