TY - BOOK

T1 - Regulated Proteolysis of Arabidopsis Argonaute1

AU - Kausika, Swathi Pranavi

PY - 2015

Y1 - 2015

N2 - 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 consist of four domains namely N, PAZ, MID and PIWI domain. The PAZ and MID domains bind small RNA 3´ and 5´ends while the PIWI domain harbors the endonucleolytic activity responsible for target RNA cleavage. 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 reduced levels of AGO1 at the membrane. In this study we use N domain as a bait in yeast two-hybrid screen to identify interactors. We found several candidates, of which many bound preferentially to AGO1 and were known to be integral or peripheral membrane proteins.Further investigations into interactors led us to discover that some of them play a role in regulation of the stability of AGO1. In particular, we concentrate on three proteins namely ATG8-Interacting protein 2 (ATI2) and Auxin Upregulated F-boxes AUF1 and AUF3. We demonstrate that ATI2 binds directly to AGO1 and acts as a cargo receptor for selective degradation of AGO1 via autophagy. In the second study on AUF1 and AUF3 we provide evidence that these proteins also influence the turnover of AGO1, perhaps particularly in response to auxin. We also show that several residues in the amino-terminal part of the N domain, the so called N coil, is required for interaction with ATI2 and AUF3, and that AGO1 mutants in these residues have substantially increased half lives in vivo. This identifies the N coil as a structural element of AGO1 required for regulated proteolysis.

AB - 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 consist of four domains namely N, PAZ, MID and PIWI domain. The PAZ and MID domains bind small RNA 3´ and 5´ends while the PIWI domain harbors the endonucleolytic activity responsible for target RNA cleavage. 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 reduced levels of AGO1 at the membrane. In this study we use N domain as a bait in yeast two-hybrid screen to identify interactors. We found several candidates, of which many bound preferentially to AGO1 and were known to be integral or peripheral membrane proteins.Further investigations into interactors led us to discover that some of them play a role in regulation of the stability of AGO1. In particular, we concentrate on three proteins namely ATG8-Interacting protein 2 (ATI2) and Auxin Upregulated F-boxes AUF1 and AUF3. We demonstrate that ATI2 binds directly to AGO1 and acts as a cargo receptor for selective degradation of AGO1 via autophagy. In the second study on AUF1 and AUF3 we provide evidence that these proteins also influence the turnover of AGO1, perhaps particularly in response to auxin. We also show that several residues in the amino-terminal part of the N domain, the so called N coil, is required for interaction with ATI2 and AUF3, and that AGO1 mutants in these residues have substantially increased half lives in vivo. This identifies the N coil as a structural element of AGO1 required for regulated proteolysis.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122138771005763

M3 - Ph.D. thesis

BT - Regulated Proteolysis of Arabidopsis Argonaute1

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -