Figure 5.

HnRNPC promotes Tau exon 10 inclusion through direct binding to U-rich tracts near Tau exons 10 and 11. (a) hnRNPC interacts with Tau pre-mRNA at the boundary between Intron 10 and Exon 11 and Intron 9 flanking Exon 10, as detected by in vitro RNA pull-down assay. In vitro transcribed RNA fragments (with biotin tag) covering the indicated regions (top diagram) around Tau pre-mRNA exons 9,10 and 11 were incubated with wild-type SH-SY5Y cell lysate. RNA fragments were then isolated using streptavidin resin. The resulted protein eluents were probed with antibody for hnRNPC. (b) RNA sequences for E10-1 and E11-2. Red indicates binding regions of hnRNPC in Tau minigene pre-mRNA. U-tract (at least 4 uridines in a row) positions are highlighted in yellow. Lowercase letters indicate intronic sequence, capital letters indicate exonic sequence. (c) Interaction between hnRNPC and Tau pre-mRNA E10-1 fragment is abolished after point mutations disrupting all U-tracts (by replacing the middle uridine with cytosine in each U-tract) in the fragment sequence, as measured by in vitro RNA pull-down assay. E10-1 m represents the E10-1 fragment with all U-tracts mutated as described above. (d) Interaction between hnRNPC and Tau pre-mRNA E11-2 fragment is abolished after point mutations disrupting all U-tracts (shown as E11-2 m). (e) The impact of hnRNPC on Tau exon 10 inclusion was partially abolished when co-expressed with Tau minigene containing U-tract mutations in either E10-1 or E11-2 (shown as TM_E10-1 m and TM_E11-2 m separately). The hnRNPC-induced Tau exon 10 inclusion was completely abolished when co-expressed with Tau minigene containing U-tract mutations in both of these fragments (shown as TM_Um). TM_WT represents the wild-type Tau minigene. Results shown are from three independent experiments.