Immunological characterization of plant polyadenylate-binding proteins

doi:10.1016/0168-9452(94)90173-2

Plant Science

Volume 99, Issue 2, 1994, Pages 161-170

https://doi.org/10.1016/0168-9452(94)90173-2 Get rights and content

Abstract

We have raised antibodies against purified 70 kDa pea cytoplasmic polyadenylate-binding protein (PABP). These antibodies recognize both the 70 kDa PABP and a nuclear, 45 kDa PABP. They do not inhibit RNA binding by the purified 70 kDa PABP and do not recognize monocot or yeast PABPs, suggesting that they recognize epitopes distinct from the RNA-binding domains in these proteins. Using these antibodies, we have studied the expression of PABPs in pea during develpment and in different tissues. We find that both the cytoplasmic and nuclear PABPs are present in all tissues, and at all stages of development examined. However, the relative proportion of these two proteins is different in different tissues, with the cytoplasmic protein predominating in leaves and the nuclear protein in roots. Our antibodies also recognize two groups of proteins that appear during discrete stages of development in pea. One of these, apparent in old tissues, may be breakdown products of the cytoplasmic or nuclear PABPs. The other, which occurs only in cotyledons, cannot be assigned a function at this time.

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Cited by (12)

Nuclear and chloroplast poly(A) polymerases from plants share a novel biochemical property
2000, Biochemical and Biophysical Research Communications
Poly(A) polymerases are centrally involved in the process of mRNA 3′ end formation in eukaryotes. In animals and yeast, this enzyme works as part of a large multimeric complex to add polyadenylate tracts to the 3′ ends of precursor RNAs in the nucleus. Plant nuclear enzymes remain largely uncharacterized. In this report, we describe an initial analysis of plant nuclear poly(A) polymerases (nPAPs). An enzyme purified from pea nuclear extracts possesses many features that are seen with the enzymes from yeast and mammals. However, the pea enzyme possesses the ability to polyadenylate RNAs that are associated with polynucleotide phosphorylase (PNP), a chloroplast-localized enzyme involved in RNA turnover. Similar behavior is not seen with the yeast poly(A) polymerase (PAP). A fusion protein consisting of glutathione-S-transferase and the active domain of an Arabidopsis-encoded nuclear poly(A) polymerase was also able to utilize PNP, indicating that the activity of the pea enzyme was due to an interaction between the pea nPAP and PNP, and not to other factors that might copurify with the pea enzyme. These results suggest the existence, in plant nuclei, of factors related to PNP, and an interaction between such factors and poly(A) polymerases.
Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase
1998, Journal of Biological Chemistry
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).
Novel Genomic Regions Linked to Ascochyta Blight Resistance in Two Differentially Resistant Cultivars of Chickpea
2022, Frontiers in Plant Science
Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon 'Ca. Liberibacter asiaticus' Infection
2013, PLoS ONE
Disease resistance in plants that carry a feedback-regulated yeast poly(A) binding protein gene
2006, Plant Molecular Biology
An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDA subunit of CPSF
2003, Plant Molecular Biology

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^†: Present address: 311 Boyce Thompson Institute, Tower Road, Cornell University, Ithaca, NY 14853, USA.

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Plant Science

Abstract

References (21)

A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation

Cell

The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation

Cell

Translation initiation requires the PABP-dependent poly(A) ribonuclease in yeast

Cell

The influence of poly(A)-binding proteins on translation of poly(A)+RNA in a cell-free system from embryo axes of dry pea seeds

Biochim. Biophys. Acta

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Anal. Biochem.

Preparation of polyclonal antibodies

Methods Enzymol.

Purification and restriction endonuclease analysis of plant nuclear DNA

Methods Enzymol.

Drosophila melanogaster poly(A)-binding protein: cDNA cloning reveals an usually long 3′-untranslated region of the mRNA, also present in other eukaryotic species

Gene

A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression

Cell

Assembly of a processive messenger RNA polyadenylation complex

EMBO J.

Cited by (12)

Nuclear and chloroplast poly(A) polymerases from plants share a novel biochemical property

Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase

Novel Genomic Regions Linked to Ascochyta Blight Resistance in Two Differentially Resistant Cultivars of Chickpea

Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon 'Ca. Liberibacter asiaticus' Infection

Disease resistance in plants that carry a feedback-regulated yeast poly(A) binding protein gene

An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDA subunit of CPSF