Extended Data Fig. 9: CircFAM120A promotes cell proliferation by regulating its parental gene translation in an IGF2BP2-dependent manner.
From: Screening for functional circular RNAs using the CRISPR–Cas13 system
a,c, KD of FAM120A mRNA by shRNAs in 293FT (a) and HT29 (c) cells. b, Stable KD FAM120A by two shRNAs in 293FT cells, confirmed by one WB experiment. d, KD of FAM120A mRNA by shRNAs inhibited cell proliferation in HT29 cells, revealed by MTT assays. e, circFAM120A KD by two gRNAs reduced FAM120A protein expression in HT29 cells, confirmed by WB of one experiment; see also three independent experiments in 293FT cells (Fig. 3c). f, Schematic of sucrose gradients used to segregate fractions in polysome profiling assays. The light fraction contains ribosome subunits and single ribosomes; the heavy fraction contains polyribosomes. g, CircFAM120A KD led to altered distribution of FAM120A mRNA on ribosomes. FAM120A mRNA in individual fractions were measured by qRT-PCR and were normalized to GAPDH. h, Stability of FAM120A protein remains unchanged after circFAM120A KD. Cycloheximide (CHX) was used to inhibit translation. Quantification of results from duplicated assays was shown underneath. i, Absolute quantification of IGF2BP2 copies per cell. Results are representative of three experiments. j, Copies of circFAM120A, linear FAM120A and IGF2BP2 protein per HT29, 293FT or HeLa cells. k, IGF2BP2 was mainly localized in the cytoplasm. Left, representative image of IGF2BP2 immunofluorescence. Right, statistics of IGF2BP2 signals in each image. n = 14 images. Data are presented as means values ± s.d. l, Absolute copies of circFAM120A and linear FAM120A associated with IGF2BP2, calculated from IGF2BP2 RIP assays in 293FT cells (Fig. 3g). m-n, Distribution of IGF2BP2 protein, circFAM120A and FAM120A mRNA in fractions of polysome profiling of 293FT cells. o, m6A promoted circFAM120A binding to IGF2BP2 in vitro, detected by NB. p, m6A enhanced the capability of circFAM120A to compete with linear FAM120A binding to IGF2BP2 in vitro. Dig-labeled linear FAM120A associated with His-IGF2BP2 was detected by NB. q, Expression of FAM120A protein was partially rescued by loss of IGF2BP2 in circFAM120A KD 293FT cells, revealed by WB. (a,c,d,g,h,m,n,o,p,q) Means ± s.d. are from three independent experiments. (a,c,d,g,o,p,q) *: P < 0.05; **: P < 0.01, two-tailed student’s t-test. (h, o, p, q) Statistics were quantified from three independent experiments processed in parallel.
