Yeast Puf3 mutants reveal the complexity of Puf-RNA binding and identify a loop required for regulation of mRNA decay

Abstract

The eukaryotic Puf proteins regulate mRNA translation and degradation by binding the 3 0 untranslated regions of target mRNAs. Crystal structure analysis of a human Puf bound to RNA suggested a modular mode of binding, with specific amino acids within each of eight repeat domains contacting a single nucleotide of the target RNA. Here we study the mechanism by which the yeast Puf3p binds and stimulates the degradation of COX17 mRNA. Mutation of the predicted RNA-binding positions of Puf3p to those found in Puf5p demonstrated that a single amino acid change in Puf3p abolished detectable binding to COX17. Since this amino acid position in both Puf3p and Puf5p is predicted to contact an adenine in the respective target RNAs, the amino acid in Puf3p must play a more critical role in promoting COX17 interaction. In contrast, an amino acid change in the third repeat of Puf3p, which interacts with the only divergent nucleotide between the Puf3p and Puf5p targets, had no effect on binding COX17. These results argue that a simple set of rules cannot reliably link specific amino acid positions with target specificity. Each of these amino acid changes in Puf3p enhanced binding to the Puf5p target HO RNA, suggesting a different mode of binding to this target. Finally, we identified an outer surface loop that was dispensable for binding but was required to promote both rapid deadenylation and subsequent decapping of the COX17 mRNA, most likely as a point of protein-protein interactions.

A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

“…Decay data were analyzed using GraphPad Prism software. Site-directed mutants of Puf3p that affect RNA binding and mRNA decay (29) are analyzed in Fig. S6.…”

“…3A) (7,12,28,29). The C(Ϫ2)A double mutant was stabilized approximately 4-fold in the same cells (half-life Ϸ17 min).…”

Targeted translational regulation using the PUF protein family scaffold

“…Studies of multiple natural and mutant PUF proteins have led to a biochemical and structural understanding of how RNA selectivity can be achieved (12,16,18,21,22). Furthermore, introducing rationally designed or selected mutations can alter specificities (12,16,18,21,23).…”

“…Mutations can be rationally designed to alter RNA specificity, based on the known structures of natural PUF-RNA complexes (12,16,18,(21)(22)(23)(24). New specificities can be obtained by transferring a segment from one protein to another, by targeted mutagenesis, or by genetic selections from protein libraries (18,23).…”

mRNA stability control: a clandestine force in normal and malignant hematopoiesis

“…50 Although stabilizing RNABPs display a high affinity for consensus sequences, 37,51,52 binding is also controlled by structural determinants. 53 Fialcowitz 54 has recently demonstrated that the ARE of the TNFa transcript can assume a hairpin structure to which a stabilizing (HuR) protein can bind but a destabilizing protein (AUF1p37) that recognizes the unfolded sequence can no longer bind. Whether external stimuli or flanking sequences prompt motifs to assume higher-order structures that differ in their affinity for binding proteins is unknown.…”