Fig. 5.

Oligonucleotide directed RNaseH cleavage assay shows that PSF protects the 5′ splice site of tau exon 10. A Radiolabeled tau wild-type (left panel) or DDPAC mutant (right panel) pre-mRNA transcripts containing exon 10 and intron 10 were incubated with RNase H and the oligonucleotide complementary to the 5′ splice site at 37°C for 20 min under splicing conditions in the presence of purified PSF (lanes 2 and 3) or control GST protein (lanes 1). After incubation, RNA was isolated, separated on denaturing gel and detected by autoradiography. Lane 2 (0 min) in both the panels contain corresponding input RNA transcripts. Tau RNA transcript and its RNase H cleavage products are illustrated in the diagram between the two panels in A, with the arrowhead indicating the cleavage site and the horizontal bar below exon10–intron 10 boundary representing the DNA oligomer that is complementary to the 5′ splice site. Bottom panel represents the quantification of data from lanes 1 and 3 in each panel, of cleavage of tau RNA as detected by autoradiography. Data are an average of four independent experiments. B RNase H cleavage assay performed with the 48 nucleotide oligomer under same conditions as described in A but with increasing amounts of purified PSF protein. Cleavage products were analyzed on denaturing gel followed by autoradiography, and efficiency of cleavage was determined by calculating the ratio of total cleavage products to the corresponding total transcript input. Top panel represents a schematic representation of the stem-loop structure with the arrowhead showing the cleavage site