Figure 5. A TLX peptide promotes miR-219 processing.

(a) Mapping p68 and Drosha-interacting domain in TLX. A schematic of TLX deletion mutants and the Drosha/p68-interacting domain (Dpi) is shown on the left. A summary of p68 and Drosha-binding results is shown on the right. (b) Deletion of TLX residues 340–359 reduced the interaction of TLX with p68 substantially. HEK293T cells were transfected with HA-tagged full-length TLX (residues 1–385) or its deletion mutants (residues 1–306, 1–340 or 1–359). Lysates were immunoprecipitated (IP) with HA antibody (αHA), then probed with p68 antibody (αp68) in western blot analysis (WB). (c) Deletion of TLX residues 340–359 reduced TLX interaction with Drosha. HEK293T cells were transfected with Flag-tagged Drosha and HA-tagged full length or deletion mutants of TLX. Lysates were IP with Flag antibody (αFlag), then probed with HA antibody (αHA). A nonspecific (ns) band in the western blot was indicated. (d,e) Expressing the Dpi peptide abolished the interaction of TLX with Drosha (d), but not the interaction of TLX with HDAC5 (e), as revealed by co-IP analysis. An empty vector (−) and a control peptide (C) were included as negative controls for the Dpi peptide. Cell lysates were IP with anti-Flag antibody, then blotted with anti-HA or anti-Flag antibody. The expression of individual proteins in the transfected cells was shown by immunoblotting as input. (f) Expression of the Dpi peptide promotes miR-219 processing. miR-219 processing was monitored using the miR-219-Glo reporter. Expressing the Dpi peptide decreased miR-219-Glo activity compared to expressing the empty vector (−) or a control peptide (C); n=3. ***P<0.001 by Student's t-test. (g–i) The levels of pre-miR-219 (h) and mature miR-219 (i), but not pri-miR-219 (g), were increased by expressing the Dpi peptide, as revealed by RT–PCR. n=4 (g); n=4 (h); n=3 (i). *P<0.05 and **P<0.01 by Student's t-test in panels (h,i). ‘n' represents experimental repeats in panels (f–i).