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

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.

Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

“…After having established that the TBSV-BS3Ng isolate forms replication vesicles on the membranes of peroxisomes in Arabidopsis , we sought to isolate and characterize the VRC proteome directly from purified peroxisomes. To do so, and taking advantage of our experience with the peroxisome isolation procedure [ 34 , 39 ], we first isolated peroxisomes from non-infected and TBSV-infected dcl2-1/dcl4-2 mutant plants. Mass spectrometry (MS) analysis of peroxisomes isolated from healthy and TBSV-infected plants revealed numerous hits corresponding to peroxisomal proteins as expected ( Supplementary Table S2 ).…”

“…The absence of replicase peptides led us to conclude that this experiment did not achieve sufficient isolation of VRCs. It is likely that the cytopathic peroxisomes ( Figure 2 ) display different biophysical properties that prevent them from being isolated by density gradient fractionation using conventional procedures [ 34 , 39 ]. In addition, the large number of host proteins present in peroxisomal fractions ( Supplementary Table S2 and [ 34 ]) may interfere with the detection of possibly small quantities of viral proteins by mass spectrometry.…”

“…High molecular weight Northern blot was performed by electrophoresis of 5 μg of total RNA on 1% agarose, HEPES pH 7.4, 6% formaldehyde gels followed by capillary transfer in SSC 20x onto Amersham HyBond N+ nylon membrane and UV crosslinked for 1 min. Low molecular weight Northern blot was performed by electrophoresis of 10 μg total RNA on 15% polyacrylamide, 0.5x TBE, 7.5 M urea gels followed by electro-transfer onto Amersham HyBond NX nylon membrane and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC chemical crosslinking [ 39 ]. miR159, miR173, TAS1 (ta-siRNA255) and snU6 were detected through DNA oligonucleotides labeled with γ- 32 P-ATP using T4 PNK (Thermo Fischer, Waltham, USA).…”

“…The P19 protein of TBSV, arguably the best-studied VSR, has been shown in heterologous systems to suppress RNAi by sequestering siRNA and preventing their loading into AGO proteins [ 34 , 38 ]. While P19 can bind siRNA duplex products of all DCL proteins in planta [ 39 ], the very high affinity for 21 nt small RNA in vivo and in vitro [ 18 , 39 , 40 , 41 ] suggests that TBSV specifically relies on sequestration of DCL4 products to suppress antiviral RNAi.…”

Plant peroxisomes at the crossroad of NO and H2O2 metabolism

“…It is known that proteins with no peroxisomal targeting signals can “piggyback” into peroxisomes by interacting with peroxisomal targeting signal (PTS)‐containing proteins (Kataya et al ). It has been demonstrated that the peanut clump virus (PCV)‐encoded P15 can be targeted into peroxisomes where it is deactivated (Incarbone et al , ) suggesting a new peroxisomal function for P15 as a regulator of the virus cycle. It has been found that the 33 kDa auxiliary replicase protein (p33) of cucumber necrosis virus (CNV), which is targeted into peroxisomes, induces necrosis because it alters the peroxisomal H 2 O 2 scavenging function (Rochon et al ).…”