Fig. 5 | Nature Communications

Fig. 5

From: LncRNA REG1CP promotes tumorigenesis through an enhancer complex to recruit FANCJ helicase for REG3A transcription

Fig. 5The alternative text for this image may have been generated using AI.

REG1CP binds to FANCJ. a Silencing of FANCJ but not DHX36 inhibited REG3A mRNA expression. b FANCJ bound to wild-type REG1CP but not a REG1CP mutant with the G-quadruplex deleted (REG1CP-ΔG4). In vitro-synthesized biotin-labelled REG1CP sense RNA (REG1CP) or antisense RNA (Antisense REG1CP) or REG1CP-ΔG4 was incubated with the immunoprecipitated FANCJ from nuclear extracts of LIM1215 cells followed by EMSA. c Binding of FANCJ to REG1CP was detected using RNA pulldown assays. Proteins co-pulled down with biotin-labelled REG1CP sense probe (REG1CP-S-probe) or REG1CP antisense probe (REG1CP-AS-probe) were analysed using western blotting. d Binding of FANCJ to REG1CP was detected using RNA immunoprecipitation (RIP) assays. RNAs co-precipitated with an antibody against FANJ from nuclear extracts of LIM1215 and HT-29 cells were analysed using qPCR. e FANCJ silencing reduced REG3A mRNA levels. Relative REG3A mRNA abundance was quantitated using qPCR in LIM1215 and HT-29 cells introduced with the control or FANCJ siRNAs. f FANCJ silencing diminished upregulation of REG3A by overexpression of REG1CP. Relative REG3A mRNA abundance was quantitated using qPCR in LIM1215 and HT-29 cells introduced with the control or FANCJ siRNAs with or without overexpressing REC1CP. g An anti-G-quadruplex antibody bound to wild-type REG1CP but not a REG1CP mutant with the G-quadruplex deleted (REG1CP-ΔG4). In vitro-synthesized biotin-labelled REG1CP or REG1CP-ΔG4 were incubated with the isotype control (rabbit IgG) or an anti-FANCJ antibody followed by analysis using EMSA. In vitro-synthesized biotin-labelled MALAT1 was included as a control. h FANCJ bound to wild-type REG1CP but not REG1CP-ΔG4. LIM1215 and HT-29 cells with endogenous REG1CP stably silenced were introduced with exogenous REG1CP or REG1CP-ΔG4. FANCJ-associated REG1CP was detected using RIP assays. n = 3 independent experiments. Data are presented as Mean ± SEM (a, d, e, f, h) or representatives (b, c, g). Statistical significance was calculated using a two-tailed t-test.

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