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.mRNA degradation is a process involving the interaction and exchange of multiple multisubunit complexes and RNA binding proteins (8). Central to mRNA degradation is the removal of the poly(A) tail (deadenylation) that is controlled by a number of proteins associating with the mRNA in a structure termed the mRNP. Principal among these factors present in the mRNP are the poly(A) binding protein (PAB1), translation initiation and termination factors, the cytoplasmic deadenylases, and the factors that bind to the mRNA and elicit alterations in the mRNA degradative rate. The processes of mRNA degradation and deadenylation and the protein complexes that are involved are highly evolutionarily conserved from Saccharomyces cerevisiae to humans.The principal pathway for mRNA degradation in yeast proceeds through several steps. First, there is an initial trimming of about 15 to 20 nucleotides (nt) of the poly(A) tail to a length of about 60 to 80 nt that is specific for each mRNA and that appears to be carried out by PAN2/PAN3, presumably a cytoplasmic process (2,19,48). This trimming requires PAB1 and the translation termination factors eRF1 and eRF3 (5, 24), and all these factors are known to associate with each other (10, 23, 24, 29). Second, the major part of deadenylation utilizes the CCR4-NOT deadenylase complex (16,48). CCR4 is the catalytic component of this complex (7, 47) and shortens the poly(A) tail of mRNA to an end point size of about 8 to 12 nt (14). Poly(A) tail shortening down to an oligo(A) form (8 to 12 A's) may lead, in turn, to the reduced ability of PAB1 to bind the poly(A) tail that may alter the translation initiation...

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

“…However, our results clearly show that deleting the eRF3-NM domain does not affect mRNA stability. Hence, our work supports the notion that in yeast the Pab1-LC domain is implicated in mRNA stability independently from eRF3, probably by modulating Pab1 packing on the poly(A) tail to control the accessibility of the deadenylases (Simon and Seraphin 2007;Yao et al 2007).…”

“…This indicates that Pab1ΔLC and eRF3ΔNM do not promote a better termination by relieving a titration of eRF3 by Pab1. Because Pab1 can oligomerize (Kuhn and Pieler 1996;Melo et al 2003), overexpression of Pab1 must promote Pab1 polymerization and association on the poly (A) tail, a phenotype also shared by the pab1ΔLC strain, thus inhibiting interaction with eRF3 (Simon and Seraphin 2007;Yao et al 2007). Overall, our data indicate that translation termination in wild-type cells is not at its maximal efficiency, possibly to tolerate recoding events implicating readthrough of a stop codon (Namy et al 2004;Dunn et al 2013).…”

“…It protects the mRNA from degradation by coating the poly(A) tail and also regulates the activity of the deadenylases complexes (Doidge et al 2012;Ezzeddine et al 2012). However, it remains unsolved if the regulation of poly(A) tail degradation by PABP involves a direct role of eRF3 (Funakoshi et al 2007;Simon and Seraphin 2007;Yao et al 2007). Finally the interaction between eRF3-PABP is also implicated in mRNA surveillance by the NMD pathway.…”

Transient CPEB dimerization and translational control

“…These studies indicate that the a-helices of the RNA recognition motifs (RRMs) could form the basis of dimer formation yet still not interfere with the RNA binding that takes place on the opposite surface of b-sheet (Jang et al 2006). In contrast, other investigators find that only the monomeric forms of RNA-binding proteins can bind RNA (Cole et al 1993), and that the RRMs are used for protein-protein recognition instead of RNA recognition (Fribourg et al 2003;Yao et al 2007). Based on these studies, dimerization/multimerization is a common feature that introduces regulatory complexity, but the biochemical result, to bind or not bind RNA, may be protein-dependent.…”

Deep mutational scanning of an RRM domain of the Saccharomyces cerevisiae poly(A)-binding protein

“…The RRM domains associate directly with the RNA molecule, while the C-terminal region is not required for RNA binding or yeast viability (Sachs et al 1987;Burd et al 1991). In addition to poly(A) binding, all Pab1 RRM domains mediate protein-protein interactions (Kessler and Sachs 1998;Yao et al 2007;Richardson et al 2012). In particular, binding of Pab1 RRM2 to the eukaryotic initiation factor 4G (eIF4G) (Kessler and Sachs 1998) is presumed to promote the formation of a closed-loop structure between the mRNA cap and the poly(A) tail (Jacobson and Favreau 1983;Wells et al 1998;Amrani et al 2008) and to stimulate mRNA translation (Tarun et al 1997;Imataka et al 1998;Park et al 2010).…”