A chloroplast-localized poly(A) polymerase
In the course of a survey and characterization of poly(A) polymerases, we identified an enzyme that is novel in many respects (1). This enzyme is distinctive in two ways. In the course of purification, this enzyme resolves into two components, both of which are needed for activity in vitro (1). One of these components (which we term PAP-III) is a ribonucleoprotein. The poly(A) polymerase acts only on RNAs associated with this ribonucleoprotein (2). More recently, we have isolated cDNAs that encode this protein, and have found that it is identical to polynucleotide phosphorylase (3). This enzyme is chloroplast-localized and plays a role in RNA turnover in this organelle (4). This observation, along with other work indicating a function of poly(A) tails in promoting RNA turnover in chloroplasts (5,6), provides a conceptual link between polyadenylation and turnover in chloroplasts. However, the simple picture that may be drawn from our in vitro studies is probably not the whole story. This is indicated by the observation that a chloroplast polynucleotide phosphorylase itself possesses a selectivity for polyadenylated RNAs in vitro (7).
Early studies indicated that a principle polypeptide component of PAP-I could be recognized by a monoclonal antibody raised against the yeast poly(A) polymerase, which led to the hypothesis that PAP-I was in fact a poly(A) polymerase (1). More recently, we have isolated cDNAs encoding PAP-I (8). The product encoded by these cDNAs possesses poly(A) polymerase activity when assayed with free poly(A), thus confirming this hypothesis. More importantly, this enzyme is capable of utilizing purified PAP-III as an RNA-binding cofactor, demonstrating that our clones actually encode PAP-I, and that PAP-I consists of a single polypeptide subunit.
1. Das Gupta, J., Li, Q., Thomson, A. B., and Hunt, A. G. (1995)
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2. Li, Q., Das Gupta, J., and Hunt, A. G. (1996) A plant poly(A) polymerase requires a novel RNA binding protein for activity. Journal of Biological Chemistry 271, 19831-19835.
3. Li, Q., Das Gupta, J., and Hunt, A. G. (1998) Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase. Journal of Biological Chemistry 273, 17539-17543.
4. Hayes,R., Kudla,J., Schuster,G., Gabay,L., Maliga,P., and Gruissem,W. (1996) Chloroplast mRNA 3' end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins. EMBO J. 15, 1132-1141.
5. Kudla, J., Hayes, R., and Gruissem, W. (1996) Polyadenylation accelerates degradation of chloroplast mRNA. EMBO J. 15, 7137-7146.
6. Lisitsky,I., Klaff,P., and Schuster,G. (1996) Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA. Proc. Nat. Acad. Sci. USA 93, 13398-13403.
7. Lisitsky, I., Kotler, A., and Schuster, G. (1998) The mechanism of preferential degradation of polyadenylated RNA in the chloroplast: the exoribonuclease 100RNP/polynucleotide phosphorylase displays high binding affinity for poly(A) sequence. J. Biol. Chem. 272, 17648-17653, 1998
8. Das Gupta, J., Li, Q., Thomson, A. B., and Hunt, A. G. (1998) Characterization of cDNAs encoding a novel plant poly(A) polymerase. Plant Molecular Biology 37, 729-734.