PAB1 self-association precludes its binding to poly(A), thereby accelerating CCR4 deadenylation in vivo

Abstract

The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.

FIG. 1.

PAB1 variants that are discussed in the paper. Residues for each domain are indicated at the top.

FIG. 2.

Multiple PAB1 defects affect CCR4 deadenylation. Pulse-chase analysis was used for determination of rates of mRNA deadenylation. All analyses utilized strain AS319 as indicated in Table 1. Northern analysis of GAL1 and MFA2pG mRNA was conducted following induction of synthesis with galactose for 8 min and shutting off of synthesis at time zero with glucose. Times are in min. The poly(A) tail lengths are indicated on the left and were determined from the known sizes of the deadenylated species: GAL1-L, 290 nt; GAL1-S, 183 nt; MFA2pG, 366 nt; pG, 188 nt; and partially deadenylated GAL1-L, 314 nt, and MFA2pG, 390 nt, as indicated in the figure. dT lanes represent the RNA sample, which had been pretreated with oligo(dT) and RNase H to remove the poly(A) tail prior to Northern analysis. (A) GAL-L; (B) MFA2pG. The asterisks in PAB1-WT for panels A and B indicate the presence of the oligonucleotide poly(A) species present at the same time as the longer poly(A) species. It should be mentioned that the GAL1 and MFA2pG mRNA syntheses are incompletely turned off by glucose, as previously observed (14), and that at the 30-min time point, it is often observed that newly synthesized mRNA with long poly(A) tails becomes present. This new mRNA does not significantly obscure the data for the original pulse of mRNA synthesis.

FIG. 3.

Yeast PAB1 can form multimers. (A) Coomassie-stained PAB1 proteins following SDS-PAGE. The PAB1 proteins (7 μg each) following purification by Flag immunoprecipitation were subjected to SDS-PAGE and were derived from strain AS319. (B) Western analysis of the PAB1-purified proteins (6 μg each) using Flag antibody following nondenaturing PAGE. PAB1 variants are shown in Fig. 1. Samples were treated with RNase A prior to separation by PAGE. Treatment with RNase I gave the same results. Other experiments indicate that RNase treatment eliminates the mRNA-PAB1 interactions that under nondenaturing conditions partially obscure the individual PAB1 multimeric species. The multimeric species are also not the result of disulfide formation between PAB1 monomers, as treatment with different reducing agents did not affect PAB1 multimerization. In lane 5, the monomeric species for PAB1-ΔRRM1 is the species second from the bottom, and its C-terminal degradation product (37) is the bottom species (see also panel A, lane 2). Numbers refer to the different oligomeric species identified for wild-type PAB1. For lane 8, PAB1 following native PAGE was incubated with radioactive 7N + 23A. Similar results as that observed in lane 8 were obtained for all other deletions in that dimers were deficient in binding poly(A), and trimers and larger oligomers were nearly entirely deficient in binding. (C) Densitometric analysis of the relative abundance of the different oligomeric species. Monomeric PAB1 was set at 1.0. The values represent the averages of the results from two different experiments, and the standard errors of the means were less than 10% in all cases.

FIG. 4.

(A) BMH cross-linking of PAB1 variants. Equivalent amounts of PAB1 variants were cross-linked in vitro with and without BMH and were detected with anti-Flag antibody following SDS-PAGE. *PAB1 represents an in vivo C-terminal truncation of PAB1 as was previously observed (37). (B) Far-Western analysis of in vitro cross-linked PAB1. Cross-linked PAB1 proteins, as depicted in Fig. 4A, lanes 1 to 12, following SDS-PAGE and transfer to nitrocellulose, were incubated with radioactive 7N + 23A RNA. (C) BMH cross-linking was conducted in vivo (43), after which PAB1 was isolated and identified by Western analysis with anti-Flag antibody. Analysis of the circular PAB1 species indicates that it consists of two related bands. These two species presumably arise from cross-linking of either C70 or C109 to C368 to generate two very similar forms. PAB1-C368S failed to form both species, and PAB1-C70S formed only one species (data not shown).

FIG. 5.

PAB1 bound to poly(A) is in equilibrium with PAB1 associating with itself. Increasing amounts (μM) of the two RNAs as indicated were incubated with PAB1 in the presence of BMH (lanes 3 to 10). Lane 1, no BMH and no RNA; lane 2, BMH without RNA. PAB1 was detected by Western analysis using anti-Flag antibody.

FIG. 6.

(A) Model of linear PAB1 bound to poly(A) and circular PAB1. The long linkers between RRM2 and RRM3 (14 amino acid residues) and RRM3 and RRM4 (25 residues) compared to RRM1 and RRM2 (9 residues) can account for the circularization of PAB1. (B) Model of PAB1 oligomerization. BMH can still cross-link PAB1 dimers when the Cys residues in either RRM1 or RRM4 are mutated, suggesting that RRM1 can interact with RRM1 and that RRM4 can interact with RRM4. How the P domain stabilizes oligomer interactions is not clear and not shown.