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).

[1]  Mark Proctor,et al.  The Solution Structure of the S1 RNA Binding Domain: A Member of an Ancient Nucleic Acid–Binding Fold , 1997, Cell.

[2]  W. Gruissem,et al.  Polyadenylation accelerates degradation of chloroplast mRNA. , 1996, The EMBO journal.

[3]  P. Klaff,et al.  Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  A. Hunt,et al.  A Plant Poly(A) Polymerase Requires a Novel RNA-binding Protein for Activity* , 1996, The Journal of Biological Chemistry.

[5]  C. Higgins,et al.  A DEAD-box RNA helicase in the Escherichia coli RNA degradosome , 1996, Nature.

[6]  W. Gruissem,et al.  Chloroplast mRNA 3′‐end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins. , 1996, The EMBO journal.

[7]  Q. Li,et al.  Characterization of a novel plant poly(A) polymerase , 1995 .

[8]  E. Hajnsdorf,et al.  Polyadenylylation destabilizes the rpsO mRNA of Escherichia coli. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. R. Kushner,et al.  Polyadenylylation helps regulate mRNA decay in Escherichia coli. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Stanley N Cohen,et al.  RNA degradation in Escherichia coli regulated by 3' adenylation and 5' phosphorylation , 1995, Nature.

[11]  Jianjun Yang,et al.  Immunological characterization of plant polyadenylate-binding proteins , 1994 .

[12]  A. Sachs,et al.  Poly(A) tail metabolism and function in eucaryotes. , 1993, The Journal of biological chemistry.

[13]  S. Cohen,et al.  The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Cao,et al.  Poly(A) RNA in Bacillus subtilis: identification of the polyadenylylation site of flagellin mRNA. , 1993, FEMS microbiology letters.

[15]  M. Macdonald,et al.  Several distinct types of sequence elements are required for efficient mRNA 3' end formation in a pea rbcS gene , 1992, Molecular and cellular biology.

[16]  G. Cao,et al.  Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Nick Proudfoot,et al.  Poly(A) signals , 1991, Cell.

[18]  M. Grunberg‐Manago,et al.  Nucleotide sequence of the pnp gene of Escherichia coli encoding polynucleotide phosphorylase. Homology of the primary structure of the protein with the RNA-binding domain of ribosomal protein S1. , 1987, The Journal of biological chemistry.

[19]  G. Karlin-Neumann,et al.  Transit peptides of nuclear‐encoded chloroplast proteins share a common amino acid framework. , 1986, EMBO Journal.

[20]  N. Chua,et al.  In vitro transcription of chloroplast protein genes. , 1986, Methods in enzymology.

[21]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[22]  Julio Montoya,et al.  tRNA punctuation model of RNA processing in human mitochondria , 1981, Nature.

[23]  A. Reiner.  Characterization of Polynucleotide Phosphorylase Mutants of Escherichia coli , 1969, Journal of bacteriology.