Abstract
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.
Highlights
We have previously described a novel plant poly(A) polymerase that consisted of two separable components [11]
We describe the further purification and characterization of PAP-III. We find that this factor consists largely or Messenger RNA 3Ј-end formation in eucaryotes is an RNA processing event involving an endonucleolytic cleavage at the polyadenylation site, followed by the addition of a polyadenylate tract to the 3Ј end of the processed RNA [1]
Our studies indicate that the reaction catalyzed by the pea poly(A) polymerase occurs while the substrate RNAs are associated with these RNA-binding proteins
Summary
We have previously described a novel plant poly(A) polymerase that consisted of two separable components [11]. The other component (PAP-III) copurified with, or included, a population of RNA-protein complexes, the RNAs of which could serve as substrates for the reconstituted poly(A) polymerase. The purified calf thymus enzyme consists of a mixture of polypeptides ranging between 57 and 60 kDa [2, 3] This enzyme can polyadenylate RNAs nonspecifically (e.g. independent of the presence of a polyadenylation signal) in the presence of Mn2ϩ [3] and acts in concert with other factors to process and polyadenylate precursor mRNAs in a poly(A) signaldependent manner [4]. Our studies indicate that the reaction catalyzed by the pea poly(A) polymerase occurs while the substrate RNAs are associated with these RNA-binding proteins. They indicate that the pea poly(A) polymerase requires that substrate RNAs be bound to these proteins to be polyadenylated
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