Publications

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties–impairment of growth and induction of defense responses–indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.

We have isolated cDNA clones encoding a novel factor (PAP-I) that is a component of a multi-subunit poly(A) polymerase from pea seedlings. The encoded protein, when isolated from appropriately engineered Escherichia coli, was active as a poly(A) polymerase, either with an associated RNA binding cofactor (PAP-III) or with free poly(A) as an RNA substrate. The latter observation indicates that PAP-I is in fact a poly(A) polymerase. PAP-I bore a striking resemblance to an as yet uncharacterized cyanobacterial protein. This observation suggested a possible chloroplast localization for PAP-I. This hypothesis was tested and found to be substantiated; immunoblot analysis identified PAP-I in chloroplast but not nuclear extracts. Our results suggest that PAP-I is a component of the machinery that adds poly(A) to chloroplast RNAs.

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).

We have purified a novel factor (PAP-III) that is a component of a multisubunit poly(A) polymerase from pea seedlings. This factor consists of one or more polypeptides with molecular masses of about 105 kDa and of a population of associated RNAs that can serve as substrates for polyadenylation. When these RNAs are separated from the 105-kDa polypeptides, polyadenylation becomes dependent upon exogenously added RNA. This RNA-dependent activity does not require the presence of a polyadenylation signal in the substrate, indicating that the activity under study is a nonspecific polyadenylation activity. One or more of the 105-kDa polypeptides could be cross-linked to the products of polyadenylation labeled with [alpha-32P]ATP and to exogenously added labeled RNAs. Cross-linking of the 105-kDa polypeptides to the products of polyadenylation was not affected by the presence of exogenously added competitors, whereas cross-linking to exogenous RNAs was diminished by excesses of RNA homopolymers. Exogenous RNAs could be polyadenylated by the combination of PAP-I + PAP-III, and this activity was diminished if the binding of the exogenous RNAs to PAP-III was prevented. We conclude from these studies that PAP-III is an RNA binding protein, that polyadenylation by the poly(A) polymerase occurs while the substrate RNAs are associated with this protein, and that the pea poly(A) polymerase can only polyadenylate those RNAs that are associated with PAP-III.

A plant polyadenylation signal consists of three distinct components: a far-upstream element (FUE) that can control utilization of several polyadenylation sites, one or more near-upstream elements (NUEs) that control utilization of each site in a transcription unit, and polyadenylation site (CSs) themselves. NUEs have previously been suggested to be related to the mammalian polyadenylation signal AAUAAA. However, many plant genes do not contain AAUAAA-like motifs near their polyadenylation sites. To better understand the nature of NUEs, we conducted a systematic analysis of the NUE for one polyadenylation site (site 1) in the pea rbcS-E9 gene; this NUE lacks an AAUAAA motif. Linker substitution studies showed that the NUE for site 1 in this gene resides in the sequence AAAUGGAAA. Single-nucleotide substitutions in this domain had modest effects on the functioning of this NUE. Replacement of part of this sequence with the sequence AAUAAA increased the efficiency of this NUE. However, alteration of nucleotides immediately 3' of the AAUAAA reversed this effect. Our results indicate that the NUE for site 1 consists of as many as 9 nucleotides, that these 9 bases do not include an element that is intolerant of single base changes, that the sequence AAUAAA can function as a NUE for site 1, and that sequences flanking AAUAAA can affect the efficiency of functioning as a NUE.