Fig. 5.

LINC01615 regulates the splicing of G6PD pre-mRNA by recruiting hnRNPA1. A The nuclear and cytoplasmic fractions of DLD1 and LoVo cells were subjected to qRT–PCR. MALAT1 was the nuclear positive control; GAPDH was the cytoplasmic positive control. B RNA-FISH performed in DLD1 cells. LINC01615 probes are red, and nuclei are stained with DAPI (1000 × magnification). C The left histogram shows the G6PD pre-mRNA/mature mRNA ratio in DLD1 cells transfected with control shRNA, LINC01615 shRNA, or cotransfected LINC01615 shRNA and hnRNPA1 siRNA. The histogram on the right shows the G6PD pre-mRNA/mature mRNA ratio in LoVo cells transfected with control vector, LINC01615 overexpression plasmid, or cotransfected LINC01615 overexpression plasmid and hnRNPA1 overexpression plasmid. D Colocalization analysis was performed with specific probes against LINC01615 and a specific antibody against hnRNPA1. Nuclei are stained with DAPI. LINC01615 probes are red; hnRNPA1 is green (1000 × magnification). E Nucleotides 401–452 of LINC01615 are in the main region that interacts with hnRNPA1. F Sequence mapping of full-length (F6) and truncated LINC01615 (F1–F5). G WB of hnRNPA1 in samples pulled down by full-length (F6) or truncated LINC01615 (F1–F5). H G6PD mRNA expression in samples pulled down by full-length (F6) or truncated LINC01615 (F1–F5). I G6PD mRNA ratio in samples pulled down by full-length (F6) or truncated LINC01615 (F1–F5). J Domain mapping of Flag-labeled full-length hnRNPA1 (P5) or truncated hnRNPA1 (P1–P4). K Antibodies against Flag were used for RIP, followed by LINC01615 qRT–PCR in DLD1 cells. L Antibodies against hnRNPA1 were used for RIP, followed by G6PD pre-mRNA qRT–PCR in DLD1 cells transfected with LINC01615 shRNA or control shRNA. M The structures of the G6PD and oligonucleotide sequences used in the pulldown experiments. The sequence from nt 35 to nt 93 of exon 12 is shown diagrammatically. Oligonucleotides 43–72 nt and 79–93 nt were used in pulldown assays. N Oligonucleotides 43–72 nt and 79–93 nt were covalently linked to adipic acid beads and incubated with nuclear extracts (150 μg of protein) from DLD1 cells overexpressing LINC01615 or control cells. The proteins were eluted and analyzed with hnRNPA1 antibody. Data are the means ± SDs (n = 3 independent experiments), *p < 0.05, **p < 0.01