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Fig. 4 | Genome Biology

Fig. 4

From: The Arabidopsis APOLO and human UPAT sequence-unrelated long noncoding RNAs can modulate DNA and histone methylation machineries in plants

Fig. 4

The UHRHF1-interacting lncRNA UPAT binds to VIM1 and LHP1 in plant cells and decoys the complex away from chromatin. A RNA immunoprecipitation (RIP) assay in Nicotiana benthamiana leaves transiently co-transformed with APOLO and translational fusions expressing GFP-VIM1 or derivatives (GFP-PHD/RING1, GFP-SRA/RING2, GFP-SRA, GFP-RING2, GFP-SPACER) under the control of the 35S-CaMV promoter. Schematic representation of VIM1 protein and derivatives tested for RIP is shown on the right. Amino acid coordinates are indicated between brackets. B, C RIP assay in N. benthamiana leaves transiently co-transformed with UPAT and GFP-UHRF1, GFP-VIM1B, or LHP1-GFPC translational fusions expressed under the control of the 35S-CaMV promoter. D–F In vitro interaction of recombinant VIM1:GFP, LHP1: and the canonical RNA-binding protein NSRa, with APOLO, UPAT, GFP mRNA, and the ASCO-interacting lncRNA ASCO. Equimolar concentrations of each RNA (for 100 ng of APOLO as a reference) were incubated with purified proteins before proceeding with a regular RIP. G–H Chromatin immunoprecipitation (ChIP)-qPCR analysis of LHP1 (in D) and VIM1 (in E) binding at the YUC2 promoter in wild-type (WT), APOLO over-expression (OE APOLO-1), and UPAT over-expression (OE UPAT) plants transiently transformed or not with GFP-VIM1. In A–H, results are expressed as a percentage of the INPUT fraction. Anti-IgG antibodies were used as a negative control. Bars represent average ± SD (n = 3 biological replicates). I Venn diagrams showing the overlap of upregulated and downregulated genes in plants constitutively expressing APOLO or UPAT vs. WT 4-day-old plants grown in standard conditions (23 °C). The p-value indicated below was calculated by hypergeometric test considering all genes annotated in Araport11. Fifty-three percent of common DEG are responsive to warmth in WT plants (Additional file 2: Table S5b)

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