Monoclonal antibodies to yeast poly(A) polymerase (PAP) provide evidence for association of PAP with cleavage factor I

Abstract

Purified yeast poly(A) polymerase (PAP) was used to produce monoclonal antibodies which recognize the enzyme in immunoblots. Epitope mapping using truncated forms of PAP and cyanogen bromide cleavage products revealed two classes of antibodies. One class (N-term) recognizes an epitope in the first 100 amino acids, and a second class (C-term) is specific for a determinant located in the last 20 amino acids of PAP. These C-terminal 20 amino acids can be removed without affecting the nonspecific poly(A) addition activity of the purified enzyme. Neither antibody inhibits the nonspecific poly(A) polymerase activity or the sequence-specific activity observed in processing extracts. The antibodies show species specificity and cannot recognize mammalian, Xenopus, or vaccinia PAP. The C-term antibodies can deplete PAP from yeast whole cell extracts, resulting in loss of poly(A) addition activity. This immunodepletion also causes a reduction in the cleavage activity which can be restored by addition of yeast cleavage factor I [CF I; Chen, J., & Moore, C. (1992) Mol. Cell Biol. 12, 3470-3481], a factor needed for both the cleavage and poly(A) addition reactions. This demonstrates that a complex of PAP and CF I exists in extracts in the absence of ATP or exogenous RNA substrate. The monoclonal antibodies against yeast PAP will be a useful tool for further study of factors required for yeast mRNA 3' end processing.

Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

“…This is certainly due to the presence of a small amount of Pap1p found in the purified fraction by immunoblotting, whereas Mpe1p was never detected in the same conditions (data not shown). Indeed, Pap1p has already been shown to be associated to purified CFI (27). As previously shown (38), a low level of cleavage activity was associated with the wild-type Mpe1p-plus CPF (Fig.…”

Posttranslational Phosphorylation and Ubiquitination of the Saccharomyces cerevisiae Poly(A) Polymerase at the S/G2 Stage of the Cell Cycle

“…Immunoprecipitation was performed with 5 mg of protein from total cell lysate using 100 l of anti-Pap1 monoclonal antibody (31) or with a control monoclonal antibody against ␤-galactosidase. Unless indicated, the antibody specific for an epitope in the C terminus of Pap1 was used.…”

Cleavage Factor II of Saccharomyces cerevisiaeContains Homologues to Subunits of the Mammalian Cleavage/ Polyadenylation Specificity Factor and Exhibits Sequence-specific, ATP-dependent Interaction with Precursor RNA

“…Resolution of these somewhat conflicting observations should come with further purification of the PF I activity. Previous studies have shown that immunodepletion of PAP from yeast processing extracts has an adverse affect on cleavage (16,29), and it would not be surprising if all of the factors were assembled into a large complex via interactions that could be easily disrupted by chromatographic treatments.…”