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. Author manuscript; available in PMC: 2007 Dec 17.
Published in final edited form as: J Biol Chem. 2003 Mar 20;278(21):18997–19007. doi: 10.1074/jbc.M301800200

Fig.8. Binding of Tra2β to Tau exon 10 increases the U1 snRNP-dependent complex formation at the 5′ splice site of exon 10.

Fig.8

A, U1 protection assays. TauEx10−11 RNA substrate (WT or N279K) was added to a 12.5-μl splicing reaction using mock- or U1 snRNP-partially depleted HeLa cell cytosolic S100, purified SR proteins, and cell lysates with overexpressed Tra2β protein. Following the incubation, the oligonucleotide complementary to the 5′ splice site of exon 10 (17) was added along with RNase H (0.4 units), and the incubation was continued for another 15 min at 37 °C. The RNA cleavage products were then analyzed by gel electrophoresis. Positions of the U1-protected pre-mRNA and the RNase H cleavage products are indicated on the right. Either SR protein mixture or transiently expressed Tra2β protein increased the formation of U1 snRNP-dependent complex when N279K mutant pre-mRNA was used (lanes 10 and 12). B, in vitro splicing of TauEx10−11 RNA substrate (WT or N279K). Mock- or U1 snRNP-partially depleted HeLa cell cytosolic S100, SR proteins, and cell lysates containing overexpressed Tra2β protein were incubated with WT or N279K mutant pre-mRNA transcript under splicing conditions. Splicing products were detected after gel electrophoresis. The asterisk indicates the position of a nonspecific cleavage product generated by hybridization of the U1 RNA-specific oligonucleotide on the tau pre-mRNA.