Isolation of Small Interfering RNAs Using Viral Suppressors of RNA Interference
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The tombusvirus P19 VSR (viral suppressor of RNA interference) binds siRNAs with high affinity, whereas the Flockhouse Virus (FHV) B2 VSR binds both long double-stranded RNA (dsRNA) and small interfering RNAs (siRNAs). Both VSRs are small proteins and function in plant and animal cells. Fusing a Nuclear Localization Signal (NLS) to the N-terminus shifts the localization of the VSR from cytoplasmic to nuclear, allowing researchers to specifically probe the subcellular distribution of siRNAs, and to investigate the function of nuclear and cytoplasmic siRNAs. This chapter provides a detailed protocol for the immunoprecipitation of siRNAs bound to epitope-tagged VSR and subsequent analysis by 3′-end-labeling using cytidine-3′,5′-bis phosphate ([5′-32P]pCp) and northern blotting.
KeywordsRNAi siRNA Endo-siRNA Immunoprecipitation Viral suppressor of RNAi B2 P19 RNA purification pCp labeling RNA detection Drosophila
We thank D. Kirschner and B. Berry for VSR expression vectors and D. Fagegaltier and A.L. Bougé for fruitful discussions. This work was supported by postdoctoral fellowships from the Agence Nationale de la Recherche to C.C. (grant number ANR BLAN 1210 01 “Nuclear endosiRNAs” to C.A.) and Ph.D. fellowships from the French government to M.vd.B.
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