Fig. 7. How(S) facilitates the splicing of stripeA minigene. (A) S-2 (Schneider) cells transfected with stripeA minigene containing the intronic sequences of stripeA flanked by partial sequences of its 3' an 5' exons, were co-transfected with different forms of How proteins. (B) The splicing of the two exons was monitored by RT-PCR using primers specific for the two flanking exons (509F and 5020R, see scheme). All RNA samples were normalized using tubulin-specific primers. In the presence of wild-type How(S) the splicing reaction was facilitated by threefold. By contrast, a mutant form of How(S) containing an NLS did not induce such elevation, nor did the nuclear-specific How(L) form. A mutant How(L), which is also cytoplasmic, did induce a mild elevation (1.5-fold). (C) Western analysis of the transfected S-2 crude extracts using anti-HA antibody to detect transfected How constructs. (D) A protein-RNA binding assay was performed on in vitro transcribed stripeA intronic RNA sequences labeled with biotin and precipitated with HA-tagged in vitro translated How(S) or mutated form of How(S), How(S)^e44, which does not bind RNA. Western analysis shows the presence of HA-tagged How(S) in the beads only in the presence of stripeA intronic sequences, while its levels are equal in all the binding reactions.