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

The eukaryotic Puf proteins regulate mRNA translation and degradation by binding the 3′ 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

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3′ untranslated regions (3′ UTRs) of target mRNAs. In yeast, Puf3p binds the 3′ UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3′ UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3′ UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3′ UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain

The Neurofibromatosis 2 Protein, Merlin, Regulates Glial Cell Growth in an ErbB2- and Src-Dependent Manner▿

Individuals with the inherited cancer predisposition syndrome neurofibromatosis 2 (NF2) develop several central nervous system (CNS) malignancies, including glial cell neoplasms (ependymomas). Recent studies have suggested that the NF2 protein, merlin (or schwannomin), may regulate receptor tyrosine kinase signaling, intracellular mitogenic growth control pathways, or adherens junction organization in non-nervous-system cell types. For this report, we used glial fibrillary acidic protein conditional knockout mice and derivative glia to determine how merlin regulates CNS glial cell proliferation. We show that the loss of merlin in glial cells results in increased proliferation in vitro and in vivo. Merlin regulation of glial cell growth reflects deregulated Src activity, such that pharmacologic or genetic inhibition of Src activation reduces Nf2−/− glial cell growth to wild-type levels. We further show that Src regulates Nf2−/− glial cell growth by sequentially regulating FAK and paxillin phosphorylation/activity. Next, we demonstrate that Src activation results from merlin regulation of ErbB2 activation and that genetic or pharmacologic ErbB2 inhibition reduces Nf2−/− glial cell Src/Src effector activation and proliferation to wild-type levels. Lastly, we show that merlin competes with Src for direct binding to ErbB2 and present a novel molecular mechanism for merlin regulation of ErbB2-dependent Src signaling and growth control