1. MED12 association with ncRNA-a is disrupted by loss of CARM1 and TDRD3.
MED12 association with ncRNA-a is disrupted by loss of CARM1 and TDRD3. (A) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d to knockdown CARM1 (see inset). Lysates were subjected to UV-RIP using IgG or MED12 antibodies and analyzed by RT–qPCR with primers specific for the indicated ncRNA-a. Error bars represent SD based on replicates (n = 3). (B) MCF-7-Tet-on-shTDRD3 cells were induced with doxycycline (1 μg/ml) for 8 d to knockdown TDRD3 (see inset). The cells were subjected to UV crosslinking followed by RNA IP (UV-RIP) using IgG or MED12 antibody and analyzed by RT–qPCR with primers specific for the indicated ncRNA-a. Error bars represent SD based on replicates (n = 3). (C) HEK293T cells transiently expressing FLAG-MED12 (WT) or FLAG-MED12R1899K (Mut) were UV crosslinked, lysed, and incubated with FLAG antibody. The immunoprecipitated RNAs were then analyzed by RT–qPCR to assess ncRNA-a levels. Inset shows similar immunoprecipitated levels of WT and Mut proteins. (D) ncRNA-a5 is located near GREB1 on human chromosome 2p25.1, along with E2F6 and ROCK2 genes. (E) Knockdown of ncRNA-a5 results in reduced expression of GREB1, as gauged by RT–qPCR. This experiment was performed in MCF-7 cells induced with E2 for 4 h prior to RNA isolation. RT–qPCR was performed using primers specific for the genes shown. Target gene expression was normalized to β-actin. Error bars represent SD based on three independent experiments. *P < 0.05, **P < 0.01, and ***P < (t test).
Donghang Cheng et al. LSA 2018;1:e
© 2018 Cheng et al.