Identification of the Mechanisms of mRNA Regulation by PUF Proteins.

Abstract

PUF proteins are conserved RNA-binding proteins that regulate mRNAs through sequence-specific binding. PUFs play integral roles in important biological processes including: development, fertility, and synaptic plasticity. Several mechanisms have been proposed for repression by PUF proteins, including: inhibition of translation initiation, elongation, and activation of deadenylation and mRNA decay. The contribution of these mechanisms was tested in S. cerevisiae, and D. melanogaster models. In yeast, Puf4p and Puf5p negatively regulate the HO mRNA, and both accelerate deadenylation through recruitment of the Pop2-Ccr4 deadenylase machinery. Surprisingly, repression by Puf4p requires deadenylation, whereas Pufp5p does not. We identified the eIF-4E binding protein, (4EBP) Eap1p, to be necessary for repression by Puf5p. Collectively, my findings show that Puf5p activates mRNA decapping, and subsequent 5′-3′ mRNA decay through association with Eap1p, and the activator of decapping, Dhh1p. I have demonstrated that Puf5p recruits Eap1p to HO mRNA to activate mRNA decapping, and this data suggests a model where Puf5p recruits Eap1p, Dhh1p, as well as the decapping and deadenylation machinery to HO mRNA. Eap1p and Dhh1p may collaborate to evict the translational machinery, including eIF-4E and eIF-4G, from a targeted mRNA; this allows the decapping enzyme to access the 5′ cap. I have identified a novel mechanism of repression via a 4EBP in the activation of mRNA decapping. The Drosophila PUF protein, Pumilio, has multiple repression domains including the conserved RNA-Binding Domain RBD. We sought to define the mechanisms of repression exerted by the RBD, and found that, like yeast Puf5p, the RBD accelerates deadenylation and degradation of target mRNAs. Acceleration of deadenylation by the RBD is catalyzed by Ccr4 and Pop2, and requires the Poly(A) Binding Protein, Pabp. Interestingly, Pabp contributes strongly to overall PUM repression, whereas deadenylation is not required for repression. We propose that the PUM RBD targets Pabp to translationally repress an mRNA, and this exposes the poly(A) tail to deadenylases recruited by PUM. Subsequently PUM elicits degradation of the target mRNA through activation of mRNA decapping. Together, this work defines the evolutionarily conserved repression mechanism of PUF proteins via deadenylation, decay and translational repression via Pabp.