Fig. 3

in vivo functionality of recombinant DCL4 in S. cerevisiae on a viral replicase-assisted DI-RNA replicon. A Schematic representation of expression cassettes used to co-express CymRSV p33/p92 and DI-RNA replicon. CymRSV RNA-dependent RNA polymerase (RdRp) was under the control of the constitutive alcohol dehydrogenase (ADH) promoter and DI-RNA was under the control of the galactose-inducible GAL1 promoter. The arrow (in red) represents the tobacco ringspot virus (TRSV) satellite RNA ribozyme (Rz) [21]. B Western blot analysis of whole cell lysates showing HA-DCL4 and/or p33 expression after 24 h growth at 21°C. Detection of HA-DCL4 was performed with a specific anti-HA monoclonal antibody. After stripping, detection of the p33 component of the CymRSV RdRP was performed on the same membrane with a specific polyclonal antiserum. (-) indicating that the corresponding empty plasmid was used for yeast transformation. C Northern blot analysis of whole cell lysates showing DI-RNA accumulation and replication. DI-RNA was detected with a specific DIG-labeled RNA probe. (−) indicates that the corresponding empty plasmid was used for yeast transformation. D Denaturing PAGE of sRNA-enriched fraction stained with EtBr from yeast cells co-expressing HA-DCL4, CymRSV p33/p92 and DI-RNA replicon (lane 2). 10 bp Ladder in lanes 1 and 3. E Size distribution of total sRNAs in cells co-expressing CymRSV p33/p92 and DI-RNA replicon in absence (dark gray) and presence (gray) of HA-DCL4. F Size distribution of DI-RNA derived sRNAs. G Distribution of the 5′-end nucleotide of sRNAs along the 477-nt long DI-3 RNA. Blue and red refer to ( +) and (−) orientation, respectively