Three-hybrid screen. A hybrid RNA construct (pRH5 0 H) containing the sequence of Pol b 3 0 UTR (from +3 to À208 downstream of the stop codon) was generated by inserting the Pol b sequence fragment into the unique SmaI site downstream of the phage MS2 sequence in the pRH5 0 vector (Invitrogen). The pRH5 0 H (H for hairpin) and cDNA library plasmids were sequentially transformed using the lithium acetate method (27) into the yeast R40C-W strain, which contains both HIS3 and b-galactosidase (b-gal) promoters integrated into its genome. Transformed yeast were plated on YC medium lacking tryptophan, uracil and histidine (YC-WUH) and containing 5 mM 3-aminotriazole (3-AT). Transformants were replicated on 25 mM 3-AT and after 3-6 days large colonies were picked for analysis. Selected transformants were screened out by the expression of beta-galactosidase using the colony lift assay with 5-bromo-4-chloro-3indolyl-beta-D-galactopyranoside (X-gal) as the substrate (CLONTECH Yeast Protocols Handbook). To eliminate RNA-independent false positives, plasmids were isolated (28) from selected blue colonies and used to transform electrocompetent E. coli KC8 cells. This bacterial strain enables complementation of yeast auxotrophy marker genes. Colonies with plasmids bearing the yeast TRP1 gene (pYESTrp1 cDNA clones) were isolated on M9 minimal medium lacking tryptophan. Plasmids were isolated, verified by restriction analysis and re-transformed into R40C-W, previously transformed with pRH5 0 H. Double transformants were checked again by the b-gal colony lift assay. This method of selection eliminated false-positive clones, leaving RNA-dependent positives. Positive clones were sequenced and analyzed by BLAST search.