Figure 4

HnRNP L interacts with PTB through proline-rich region (PRR) to synergistically repress inclusion of exon P3A.

(a) Schematic domain-structures of hnRNPs L and LL and their mutant derivatives. Interaction of each protein product with PTB is indicated on right according to the results from panels (d) and (e). RRM, GRR and ND indicate RNA recognition motif, glycine-rich region and not detected respectively. (b) RT-PCR of pSPL3-MS2 products after the indicated MS2-tagged trans-acting effectors are introduced into SH-SY5Y cells. Bars and lines represent mean and SD, respectively, of three independent experiments. Immunoblotting shows expression of effectors in the nuclear lysate of SH-SY5Y cells. (c) Schematic binding sites of hnRNP L and PTB in exon P3A and upstream PPT (YYYY) respectively. (d) Interaction of PTB with His-tagged hnRNP L and its indicated mutant. Nuclear extract of transfected SH-SY5Y cells is immunoprecipitated with anti-His antibody and assayed for interaction with endogenous PTB by immunoblotting (IB). Open arrowheads point to IgG heavy chain (HC) that was non-specifically precipitated. (e) Interaction of PTB with His-tagged hnRNP LL and its indicated mutant. See (d) for the procedures and other labels.