The RRM1 domain of the poly(A)-binding protein from Saccharomyces cerevisiae is critical to control of mRNA deadenylation

Abstract

The poly(A)-binding protein PAB1 from the yeast Saccharomyces cerevisiae plays an important role in controlling mRNA deadenylation rates. Deletion of either its RRM1 or proline-rich domain (P domain) severely restricts deadenylation and slows mRNA degradation. Because these large deletions could be having unknown effects on the structure of PAB1, different strategies were used to determine the importance of the RRM1 and P domains to deadenylation. Since the P domain is quite variable in size and sequence among eukaryotes, P domains from two human PABPCs and from Xenopus were substituted for that of PAB1. The resultant PAB1 hybrid proteins, however, displayed limited or no difference in mRNA deadenylation as compared to PAB1. In contrast to the P domain, the RRM1 domain is highly conserved across species, and a systematic mutagenesis of the RRM1 domain was undertaken to identify its functional regions. Several mutations along the RNA binding surface of RRM1 inhibited deadenylation whereas one set of mutations on its exterior non-RNA binding surface shifted deadenylation from a slow distributive process to a rapid processive deadenylation. These results suggest that the RRM1 domain is the more critical region of PAB1 for controlling deadenylation and consists of at least two distinguishable functional regions.

Global Analysis of mRNA Isoform Half-Lives Reveals Stabilizing and Destabilizing Elements in Yeast

“…Pab1 binding to poly(A) tails in vivo requires a minimum stretch of ~10–12 consecutive A residues (Sachs et al, 1987) and is highly cooperative. Pab1 binding is implicated in the inhibition of poly(A) tail deadenylation by the Ccr4-Pop2 complex that eventually results in the exonucleolytic degradation of mRNAs by either the exosome or the decapping/Xrn1 complexes (Simón and Séraphin, 2007; Zhang et al, 2013). …”

Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

“…To selectively purify specific types of translating complexes, we conducted a one-step Flag agarose purification of our complexes prior to our AUC analyses using strains containing different Flag-tagged translational factors [ 11 ]. The Flag-tagged factors studied were Flag-PAB1, eIF4E-Flag, RPL25A-Flag (component of the 60S ribosome), and Flag-SBP1 [ 11 , 12 , 15 ]. Each of these purifications would purify different pools of ribosomal-associated material.…”

“…Each of these purifications would purify different pools of ribosomal-associated material. The 80S free ribosome would be purified using RPL25A-Flag [ 11 , 14 , 16 ], whereas Flag-PAB1 purified translating complexes [ 11 , 15 , 17 , 18 ] containing mRNA with poly(A) tails of at least 24 A’s [ 19 ], the minimal size to which PAB1 binds [ 20 ]. eIF4E-Flag and Flag-SBP1 would purify complexes containing eIF4E and SBP1, respectively, irrespective of whether the translational complexes contained a poly(A) tail.…”

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression

“…Consistent with our results, there is a report that PAIP2 knockdown and PABP overexpression diminished miRNA function, and the elevated levels of endogenous PAIP2 enhanced miRNA efficacy ( 10 ). However, whether PABP stabilizes mRNA adenylation is still controversial, and there are contradicting reports that PABP is required for CCR4-mediated deadenylation ( 46 – 48 ).…”