Figure 6.
CARM1 is enriched at the NEAT1 promoter and inhibits NEAT1 transcription and paraspeckle formation. (A) CARM1 deficiency promotes the expression of both isoforms of NEAT1. (B) CARM1 knockdown increases paraspeckle formation. (Left) Colocalization of NEAT1 RNA (green) and p54nrb protein (red) revealed paraspeckles in scramble and CARM1 knockdown (KD) HeLa cells. (Right) Statistical analysis of numbers of paraspeckles from >300 cells in scramble and CARM1 knockdown HeLa cells. The P-value from a one-tailed t-test in the pairwise comparison is shown. (C) CARM1 knockdown increases paraspeckle formation in HEK293 cells. (D) CARM1 is enriched on the promoter region of NEAT1. CARM1 ChIP followed by qPCR analyzed the occupation of CARM1 on the NEAT1 gene. The positions of examined primers are shown in E. (E) CARM1 deficiency promotes the transcription of nascent NEAT1. (Top) A schematic drawing indicates the primer sets used. (Bottom) A crude preparation of nuclei was subjected to NRO assays under the indicated conditions in HeLa cells. Nascent transcription of NEAT1 detected from scramble nuclei was defined as one. In A, D, and E, error bars represent ±SD in triplicate experiments. (*) P < 0.05; (**) P < 0.01; n ≥ 3.