Fig. 2: mOct4P4 deletion analysis identifies the minimal RNA regions essential for lncRNA function.

a Schematic representation of the mOct4P4-24xMS2 deletion constructs. Gray boxes, sequences with homology to Oct4/OCT4 5′UTR; gray lines, sequences with homology to Oct4/OCT4 3′UTR; 334 bp lines represent centrally located, spliced fragment present in mOct4P4. 24xMS2 RNA stem loop motifs located at the 3′ end of mOct4P4 deletion constructs are indicated. Arrows indicate the locations of RT-PCR primers used to amplify mOct4P4 deletion constructs. b Levels of ectopic expression of mOct4P4-24xMS2 deletion construct (a) in mESCs, as determined by qRT-PCR. p Values relate to CTRL. c Subcellular localization of lncRNAs derived from mOct4P4-24xMS2 lncRNA deletion constructs (a) in mESCs, as determined by qRT-PCR. Expression values are shown as percentage of total RNA levels. p Values indicate significant nuclear versus cytoplasmic localization. d Oct4 mRNA levels in mESCs with ectopic expression of mOct4P4 deletion constructs (a), as determined by qRT-PCR. Expression values were normalized against gapdh. p Values relate to CTRL. e Representative image of OCT4 western blotting analysis in mESC cells overexpressing mOct4P4 deletion constructs shown in (a). ACTIN was used as loading control. Numbers represents values of OCT4 expression as mean of three independent experiments (control was set “100”). f, g anti-flag ChIP (f) and anti H3K9me3 ChIP (g) on the Oct4 promoter region in mESCs stably overexpressing 24xMS2 tagged deletion constructs shown in (a). qRT-PCR was performed to measure promoter enrichment; p values relate to MS2-flag/24xMS2-CTRL. Precise p values are indicated; n number of independent experiments carried out.