Fig.2. N279K mutation enhances exon 10 splicing.

A, alternative splicing of exon 10 from transfected WT or N279K mutant tau minigene. The tau minigene constructs were transfected into HeLaRB, HEK293, and N2a cells. The splicing products expressed from the transfected minigene were detected by RT-PCR with primers specific to the transfected tau minigenes. The positions of exon 10-containing and exon 10-skipping splicing products are as indicated. B, in vitro splicing of TauEx9−11d5 (WT or N279K) (lanes 1−6), TauEx9−10d5 (WT or N279K) (lanes 7 and 8), and TauEx10−11d5 (WT or N279K) (lanes 9 and 10) RNA substrates. ³²P-Labeled pre-mRNA substrates were incubated in HeLa nuclear extracts under the splicing conditions. The reactions shown in lanes 3−6 contained different amounts of the exogenous purified U2AF65 protein. Positions of pre-mRNA and splicing products are as indicated. C shows the quantification using a PhosphorImager of splicing products expressed as the ratio of exon 10-containing mRNA (Ex10+) to exon 10-skipping mRNA (Ex10−) in splicing reactions with TauEx9−11d5 substrate (lanes 1−6). In the absence of exogenous U2AF65, the HeLa nuclear extract did not produce a sufficient amount of exon 10-containing splicing products that were detectable by the PhosphorImager; therefore, quantification was only carried out for splicing reactions containing the supplemented U2AF65 protein.