PhD defense: Swathi Kausika

Regulated proteolysis of Arabidopsis ARGONAUTE1

Supervision
Peter Brodersen, Associate Professor, University of Copenhagen

Exam Committee
Jeppe Vinther, Associate Professor, University of Copenhagen
Daniel Hofius, Professor, Department of Plant Biology, Sveriges lantbruksuniversitet
Sébastien Pfeffer, Research Director, Institut de Biologie Moléculaire et Cellulaire du CNRS

Abstract
Argonaute (AGO) proteins are key effectors of RNA Induced Silencing Complex (RISC) that mediates RNA interference or gene silencing. AGO binds small RNA and uses base pairing to small RNA to bring about repression of specific, complementary target RNA by cleavage or translational repression. These are large multi domain proteins found in both eukaryotes and prokaryotes. They constitute four domains namely N, PAZ, MID and PIWI domain and have assigned key functions. The PAZ and MID domains bind small RNA 3´ and 5´ends while the PIWI domain harbors the slicer activity. This study focuses on the function of poorly characterized N domain. Arabidopsis thaliana AGO1 is a peripheral membrane protein and membrane association is important for function. Previous studies in the model plant showed that mutation in the N domain resulted in alleviated levels of AGO1 at the membrane. Therefore, we hypothesized that the N domain could play a role in the recruitment of AGO1 to the membranes. In this study we use N domain as a bait in yeast two-hybrid screen to identify interactors. We found a diverse list of candidates that preferentially bound to AGO1 and some of the candidates lost interaction with the N domain mutant supporting its role in membrane recruitment. Further investigations into interactors led us to discover that some of them could act as potential binding partners or signaling receptors for AGO1 and regulate its stability. In particular, we concentrate on three proteins namely ATI2, AUF1 and AUF3. We demonstrate that Atg8-interacting protein ATI2 binds directly to AGO1 and acts as a cargo receptor for selective degradation of AGO1 via autophagy. In the second study involving auxin-upregulated F-box proteins AUF1 and AUF3 we provide evidence for auxin mediated turnover of AGO1. Lastly, the versatile list of positive interactors illustrates that N domain is a functional binding platform essential for AGO1 regulation.