The protein kinase TOUSLED facilitates RNAi in Arabidopsis

RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interferenc

Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 protein

BM benefited from an IdEx postdoctoral fellowship from the Université de Strasbourg. Financial support to MI was provided in part by the INTERREG V Upper Rhine program Vitifutur, Transcending borders with every project. Work in the JT laboratory is funded by the U.K. Biotechnology and Biological Sciences Research Council (BB/M007200/1).Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.Publisher PDFPeer reviewe

APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

Background: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed

La bataille du silence

Le RNA silencing est un mécanisme opéré par de petites molécules d’ARN qui inhibent l’expression de gènes tant au niveau transcriptionnel que post-transcriptionnel via des interactions séquence-spécifiques. Conservé chez la plupart des eucaryotes, ce phénomène est impliqué dans le maintien de l’intégrité des génomes ou dans la régulation de facteurs spécifiques au cours du développement, aussi bien chez les animaux que chez les plantes. Chez ces dernières, le RNA silencing joue également un rôle crucial dans la défense antivirale. Pour contrer cette réponse, les virus ont développé une batterie de stratégies pour inhiber ce mécanisme. Cette revue détaille les mécanismes du RNA silencing et de son inhibition au cours des interactions plante/ virus et suggère les possibles conséquences de cette bataille moléculaire sur l’évolution de leur génomes respectifs

Etude des stratégies mises en oeuvre par le Peanut clump virus pour assurer l'amplification de son génome (rôle de la protéine P15)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

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

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

Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5′-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas