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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.
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