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)
Characterization of a novel plant polyadenylate polymerase. Plant Sci.110,
215-226.
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.