Mechanisms of mRNA Regulation by Pumilio and Nanos.

Abstract

Drosophila Pumilio and Nanos proteins function together to control development, germline stem cell maintenance, and neurological functions. How Pumilio and Nanos collaborate is not completely understood. Pumilio belongs to a conserved eukaryotic family of RNA binding proteins that bind with high affinity and specificity to Pumilio Response Elements (PREs), consequently inhibiting protein synthesis. Nanos belongs to a conserved family of tandem zinc finger proteins. Together they are implicated in mRNA localization, translational inhibition, and decay.

To investigate how Pumilio and Nanos work together, I developed cell-based and biochemical assays that measure their repression and RNA binding activities. Surprisingly, I found that Pumilio represses PRE-containing mRNAs independent of Nanos. I identified three unique domains in Pumilio capable of inhibiting protein expression. Two domains responsible for auto-regulation of repression were also discovered. I characterized multiple mechanisms of Pumilio-mediated repression, including interference of poly-adenosine binding protein and acceleration of deadenylation.

While Nanos is not necessary for regulation, I found that it robustly enhances Pumilio-mediated repression by two mechanisms. First, Nanos complexes with Pumilio and increases the affinity of Pumilio for PRE containing RNA. Nanos even confers Pumilio binding to RNAs that are not normally recognized. This effect is mirrored in cells; Nanos stimulates Pumilio-dependent repression of mRNAs bearing weak/degenerate PREs. Second, we discovered that Nanos possesses a repression domain that synergistically promotes repression with Pumilio. Together, these data reveal a new mechanism of Nanos repression that contributes to combinatorial control by Pumilio.

I propose that Nanos augments the spatiotemporal control of mRNAs by Pumilio; Nanos specifies how tightly Pumilio binds mRNA and tunes the level of repression. The auto-regulatory and repression domains within Pumilio help to ensure a specific response. These findings advance our understanding of how Pumilio and Nanos homologs might dynamically control targets in the germline and the nervous system.