Figure 2.
TDP-43 binds intron 9 of tau pre-mRNA. A, various amounts of pCI/TDP-43 were co-transfected with pCI/SI9-SI10 into HEK-293FT cells. The alternative splicing products of tau exon 10 were analyzed by RT-PCR. The expression of TDP-43 tagged with HA was detected by Western blotting. The ratio of inclusion/exclusion of tau exon 10 was plotted against the amount of pCI/TDP-43. B, flow chart of the assay of TDP-43 binding to tau pre-mRNA. C, degree of match of pre-mRNA sequence of SI9-SI10 with TDP-43 in binding assay. SI9-SI10 was transcribed in vitro into pre-mRNA. The pre-mRNA was incubated and UV-cross-linked with immunopurified TDP-43 from HEK-293FT cells. The unbound RNA was then hydrolyzed with RNase, and TDP-43-bound RNA fragments were sequenced. The x axis is the map site value; the y- axis is the distribution coefficient. D, diagram showing the binding sites of the pre-mRNA of tau mini-gene SI9-SI10 with TDP-43 determined from B. A core binding region, nt 298–438, contained a core binding site, nt 331–425, flanked by two binding sites, nt 298–346 and 390–438. There were other binding sites, including nt 639–687, 1240–1288, 1341–1389, and 1845–1894. E, pCI/SI9-LI10 was co-transfected with pCI/TDP-43·HA into HEK-293FT cells. RIP was carried out with anti-HA. Co-immunoprecipitated tau SI9-LI10 pre-mRNA was reverse transcribed into cDNA and amplified with two sets of primers against the core region of tau intron 9 to obtain 198- and 295-bp PCR products. The RT-PCR products were separated by agarose electrophoresis and quantitated by densitometry. The data are presented as mean ± S.D. (error bars) (n = 3). **, p < 0.01. F, pCI/SI9-SI10 or its deletion mutants, pCI/SI9-SI10Δ298–438 and pCI/SI9-SI10Δ331–425, were transfected alone or together with pCI/TDP-43·HA into HEK-293FT cells. RIP was carried out 48 h after transfection. Immunoprecipitated tau SI9-SI10 pre-mRNA was determined by RT-PCR with set 2 primers. The expression of TDP-43 in the input was determined by Western blotting with anti-HA.