Fig. 3

LINC02273 interacts with hnRNPL. a Identification of proteins associated with LINC02273. Up: silver staining of RNA pull-down proteins. Whole cell lysates and nuclear fraction were incubated with in vitro transcribed RNA of sense (S) and anti-sense (AS) LINC02273. LINC02273-S enriched bands were analyzed by MS (bands in dash lines), n = 3 independent experiment. LIN02273-AS was used as negative control. Bottom: Validation of LINC02273 and hnRNPL interaction by immunoblotting. b RIP and RT-qPCR were shown. IgG was used as negative control. n = 3, biological replicates. c Schematic view of truncated LINC02273. FL (full length)-AS was used as negative control. RNAs corresponding to indicated fragments were biotinylated and incubated with whole cell lysates and subjected to streptavidin pull-down. d hnRNPL binding domain on LINC02273. Up, immunoblotting was used to confirm hnRNPL binding. Bottom: Input RNA (3 μg) was shown by native agarose electrophoresis. e Schematic view of RNA binding motif of hnRNPL. Up, schematic view of hnRNPL ORF and four RRM domains. Bottom, hnRNPL was truncated according to RRMs. f RNA pull-down was performed with ectopically overexpressed truncated hnRNPL with 3 × FLAG in 293 T cells and detected by immunoblotting. n = 3, biological replicates. g RNA expression of LINC02273 after hnRNPL KO. h Half-life of LINC02273 in WT and hnRNPL KO MDA-MB-231 cells treated with 5 μg/mL Actinomycin D. c-myc served as positive control. RNA was extracted and assayed by RT-qPCR. n = 3, biological replicates. i LINC02273 stability assay. LINC02273 with or without CA-repeat was ligated to the 3′ of luciferase ORF on pmirGLO vector. hnRNPL was knocked down by pooled siRNA. RNA was assayed by RT-qPCR. n = 3, biological replicates. For g-h, RT-qPCR data are normalized to GAPDH. For (b) and g-i, means ± s.e.m. are shown, and P values were determined by independent sample t-tests. *P < 0.05, **P < 0.01, ***P < 0.001