Peroxisomal Targeting as a Sensitive Tool to Detect Protein-Small RNA Interactions through in Vivo Piggybacking.

Marco Incarbone,Christophe Ritzenthaler,Patrice Dunoyer

doi:10.3389/fpls.2018.00135

Abstract

Peroxisomes are organelles that play key roles in eukaryotic metabolism. Their protein complement is entirely imported from the cytoplasm thanks to a unique pathway that is able to translocate folded proteins and protein complexes across the peroxisomal membrane. The import of molecules bound to a protein targeted to peroxisomes is an active process known as ‘piggybacking’ and we have recently shown that P15, a virus-encoded protein possessing a peroxisomal targeting sequence, is able to piggyback siRNAs into peroxisomes. Here, we extend this observation by analyzing the small RNA repertoire found in peroxisomes of P15-expressing plants. A direct comparison with the P15-associated small RNA retrieved during immunoprecipitation (IP) experiments, revealed that in vivo piggybacking coupled to peroxisome isolation could be a more sensitive means to determine the various small RNA species bound by a given protein. This increased sensitivity of peroxisome isolation as opposed to IP experiments was also striking when we analyzed the small RNA population bound by the Tomato bushy stunt virus-encoded P19, one of the best characterized viral suppressors of RNA silencing (VSR), artificially targeted to peroxisomes. These results support that peroxisomal targeting should be considered as a novel/alternative experimental approach to assess in vivo interactions that allows detection of labile binding events. The advantages and limitations of this approach are discussed.

Highlights

Peroxisomes are small eukaryotic organelles that specialize in oxidative metabolic reactions (Kaur et al, 2009)
Previous experiments conducted with the viral suppressors of RNA silencing (VSR) P15 have shown that 21nt micro RNA (miRNA) could be detected in isolated peroxisomes, they were below detection level in northern analysis following IP experiments (Incarbone et al, 2017; MI and PD unpublished observations)
Whereas small RNA piggybacking in peroxisomes was assessed in wild-type P15-expressing plants, IP relied on a 2xFlag2xHA epitope-tagged version of this VSR (P15FHA), which may exhibit an altered small RNA-binding ability compared to the untagged P15

Summary

Introduction

Peroxisomes are small eukaryotic organelles that specialize in oxidative metabolic reactions (Kaur et al, 2009). The chemical reactions carried out within peroxisomes reflect their function and are determined by their enzymatic content, which in turn depends on species, cell type, and environmental conditions (LanyonHogg et al, 2010). All peroxisomes perform the vital task of detoxifying reactive oxygen species. Plant peroxisomes play a pivotal role in a wide range of pathways such as lipid metabolism, photorespiration, nitrogen metabolism, hormone synthesis and plant-pathogen interactions (Hayashi and Nishimura, 2003; Kaur et al, 2009). Mutations abolishing peroxisome biogenesis are embryo-lethal in Arabidopsis thaliana (Schumann et al, 2003; Fan et al, 2005)

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Conclusion