Multiple forms of poly(A) polymerases in human cells.

Abstract

We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coil. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% simlarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. col, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100-and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP.Subsequent mutational analysis revealed that both the N-and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.

Inositol 1,4,5-Triphosphate Receptor-binding Protein Released with Inositol 1,4,5-Triphosphate (IRBIT) Associates with Components of the mRNA 3′ Processing Machinery in a Phosphorylation-dependent Manner and Inhibits Polyadenylation

“…In agreement with this hypothesis, a ternary complex containing PAP, IRBIT, and Fip1 was observed in vitro. The presence of functional PAP in the cytoplasm (26,27), where IRBIT and Fip1 are likely to interact, supports the idea that a transient complex containing these three molecules can form also in vivo.…”

“…Vitro-Besides its role in the polyadenylation of nuclear RNAs, functional PAP is also present in the cytoplasm (26,27), where Fip1 and IRBIT presumably interact. Because Fip1 binds to PAP in vitro (11) and also interacts with IRBIT, we examined whether Fip1, IRBIT, and PAP could form a ternary complex.…”

Deregulation of Poly(A) Polymerase Interferes with Cell Growth

“…One of the short forms of PAP mRNA (1,12,40), PAP III, is expressed at the mRNA level in DT40 cells. Although the short PAPs, when produced as recombinant proteins, are not active in polyadenylation (40) and are not detected at the protein level in HeLa cells (35,40) or frog oocytes (1,12), the short PAPs or their mRNAs might have some function that is essential for viability. For example, we suggested previously that the short forms are the products of autoregulation (40), and perhaps this regulation is essential.…”

Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia

“…In a general sense, these increases in abundance may be due to increased usage of the respective poly(A) sites, such that more of the transcriptional output is directed toward proximal polyadenylation. Several of the activities of individual polyadenylation complex subunits are affected by phosphorylation, methylation, calmodulin, disulfide remodeling, and ubiquitin (Jacob and Rose, 1984;Thuresson et al, 1994;Colgan et al, 1996;Mizrahi and Moore, 2000;Mouland et al, 2002;Delaney et al, 2006;Ryan, 2007;Addepalli and Hunt, 2008;Ryan and Bauer, 2008;Addepalli et al, 2010;Martin et al, 2010). In addition, alterations in the activities of individual poly(A) complex subunits are often accompanied by large-scale shifts in poly(A) site choice (Jenal et al, 2012;Martin et al, 2012;Thomas et al, 2012;Duc et al, 2013;Li et al, 2015).…”