Isolation of small interfering RNAs using viral suppressors of RNA interference

Abstract

The tombusvirus P19 VSR (viral s uppressor of R NA 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 N uclear L ocalization S ignal (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′ _32 P]pCp) and northern blotting.