Fig. 1.

eaRNA^neCtnnb1maintains the transcription ofCtnnb1and facilitates the interaction of neCtnnb1 andpCtnnb1. (A–B) Transcripts derived from neCtnnb1 and Ctnnb1 in forebrains at indicated time points are shown. (B) indicates enlarged regions of neCtnnb1 (green shaded) and the Ctnnb1 (blue shaded). Please note that eaRNA^neCtnnb1 consists of three exons and is transcribed at the same direction as Ctnnb1. The sequence homology among vertebrates is shown at the top, while the Ribo-seq signal of E14.5 forebrains is displayed at the bottom. RNA-seq data were obtained from ENCODE (The Encyclopedia of DNA Elements) and the Ribo-seq data were retrieved from the GEO database (GSE169457). (C) qRT-PCR showing relative mRNA levels of eaRNA^neCtnnb1 and Ctnnb1 in Neuro-2a cells transfected with shRNAs with scrambled (Scr) sequence or against eaRNA^neCtnnb1 for 72 hours. n = 3 independent experiments. (D) Neocortical neurospheres were transfected with lentiviruses expressing indicated shRNAs and human CTNNB1 for seven days followed by cell counting. n = 3 independent experiments. (E) In the RNA-tethering experiments, indicated transcripts were attached with gRNA to target the promoter of Ctnnb1. 72 hours after transfection, the expression levels of Ctnnb1 in Neuro-2a cells were measured by qRT-PCR. n = 3 independent experiments. (F) Relative luciferase activity of Neuro-2a cells transfected with plasmids expressing BoxB-tagged LacZ or eaRNA^neCtnnb1 along with Gal4-kN and UAS-TK-Luciferase for 24 hours. n = 3 independent experiments. (G) In the 3C experiments, the relative association strength between indicated sites with the promoter of Ctnnb1 were measured in Neuro-2a cells. The test primer T3 is located within the neCtnnb1 region. Quantification data are shown as means ± SEM, statistical significance was determined using one-way ANOVA analysis (D–E) and an unpaired two-tailed Student's t-test (C and F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. ns, not significant.