Fig. 5: NAS1 and PTBP1 cooperatively enhance the IRES function of NR2F1-5′UTR. | Nature Communications

Fig. 5: NAS1 and PTBP1 cooperatively enhance the IRES function of NR2F1-5′UTR.

From: Long non-coding RNA NR2F1-AS1 induces breast cancer lung metastatic dormancy by regulating NR2F1 and ΔNp63

Fig. 5

a PTBP1 pulldown by NAS1 RNA in MCF10CA1h lysates. A schematic of the RNA pulldown assay was shown on the left. b NAS1 RNA level in RIP assays for PTBP1-interacting RNAs in MCF10CA1h expressing PTBP1-HA. c NR2F1 protein level after PTBP1 knockdown in NAS1-overexpressing MCF10AT (left) or after PTBP1 overexpression in CA1h-P2 with NAS1 knockdown (right). d NR2F1 mRNA level in RIP assay for PTBP1-interacting RNAs in MCF10CA1h expressing PTBP1-HA. e RIP assays with the MS2–GFP system to analyze the RNA–RNA interaction between NAS1 and NR2F1. A schematic of the assay was shown on the left. f RIP analyses of the binding of PTBP1 to NR2F1 mRNA in CA1h-P2 with NAS1 knockdown (n = 3 replicates from one experiment). g RIP analyses of RNA–RNA interaction between NAS1 and NR2F1 in MCF10CA1h with PTBP1 knockdown. The ratios of NR2F1 to NAS1 were shown (n = 3 replicates from one experiment). h Schematic of the pGF plasmid and the NR2F1-5′UTR segments for IRES activity analyses. Numbers in parentheses indicate the start and end base-pair positions of the segments. GFP and firefly luciferase are transcribed by the same CMV promoter, while luciferase translation is dependent on the IRES activity of the insert sequence between the two genes. i IRES activity of NR2F1-5′UTR was analyzed by a dual-luciferase reporter assay of the pGF system (n = 3 wells from one experiment). EMCV IRES was used as a positive control. NC, negative control. j PTBP1 binding sites on NR2F1-5′UTR predicted by catRAPID (top), and NAS1 binding sites on NR2F1-5′UTR predicted by IntaRNA (bottom). k RIP analyses of the binding of NAS1 to different regions of NR2F1 mRNA. l RNA pulldown assays for interaction between PTBP1 and regions of NR2F1 mRNA. m Effect of PTBP1 on IRES activity of NR2F1-5′UTR-PT (n = 3 wells from one experiment). n Effect of NAS1 overexpression on IRES activity of NR2F1-5′UTR (n = 3 wells from one experiment). o Schematic of the mechanism by which PTBP1 and NAS1 regulate the IRES activity of NR2F1-5′UTR. PTBP1-mediated NR2F1 translation was halted by the GC-rich segment (i) and could be activated by the binding of NAS1 (ii). TSS transcription start site. Data represent mean ± SD. Statistical significance was determined by a two-tailed unpaired t test. Experiments were repeated at least three times independently with similar results; data from one representative experiment are shown.

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