Fig 2: Expression of a LIN28 sponge construct alters gene expression in mouse embryonic stem cells(A) Binding sites that are shared before and after sponge expression. The heatmap compares the sites detected in condition M (vertical axis) with the sites in condition N (horizontal axis).(B) Distribution of ratios that reflect changes in read density at all detected binding sites in response to the expression of a LIN28 sponge construct.(C) Cumulative distribution functions showing the minimum free energy of the ensemble for all binding sites detected before sponge expression (green; n= 992 sites) and after sponge expression (red; n = 70 sites) compared with randomly chosen sequences outside of eCLIP-identified binding sites (dashed gray).(D) Design of gene expression constructs used to track changes in let-7 miRNA activity in response to the depletion of LIN28 by sponge in single cells, as measured by analytical flow cytometry. Differences in miRNA activity are measured via changes in turquoise-to-cherry fluorescence as a function of sponge expression (citrine). All fluorescence data are binned, and trends map the average response to LIN28B expression (N = 3, ± SEM).(E) Changes in polysome enrichment in V6.5 mESCs and Dicer knockout mESCs after the expression of LIN28 sponge.(F) A rolling mean plot showing the relationship between the change in polysome enrichment (rank ordered from the most repressed to the most activated by sponge construct) and the frequency of let-7 target sites per gene.

Fig 3: LIN28B inhibits p53 protein but not mRNA level independent of MDM2/4.A Representative western blots of p53 and LIN28B expression in A2780 and TOV-112D cells with LIN28B knockdown (shLIN28B) and overexpression (LIN28B) from three independent experiments. B Representative western blots of p53 and LIN28B expression in LOXI and CRMM2 cells with LIN28B knockdown (shLIN28B) from three independent experiments. C Correlation of LIN28B and p53 protein expression in ovarian cancer cell lines (n = 29) based on Supplementary Fig. 3 and our previous publication [23]. D Representative immunohistochemistry staining image of p53 in LIN28B knockdown (shLIN28B) and overexpression (LIN28B) xenograft tumors. Bar, 100 μm. E Representative western blots of MDM2 and MDM4 expression in A2780 and TOV-112D cells with LIN28B knockdown (shLIN28B) and overexpression (LIN28B) from three independent experiments. F qPCR of TP53 mRNA level in LIN28B knockdown (shLIN28B) and overexpression (LIN28B) samples. Ctrl = control. Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant. G, qPCR of TP53 mRNA in LIN28B knockdown (shLIN28B) and overexpression (LIN28B) cells treated by actinomycin D (10 μg/ml). Representative data of three independent experiments (mean ± s.e.m.). n.s. = not significant.

Fig 4: LIN28B interacts with TP53 mRNA at the 5′ untranslated region (UTR).A Schematic diagram of RNA immunoprecipitation (RNA-IP) and qPCR detection of TP53 mRNA enrichment in A2780 and TOV-112D cells using isotype control IgG or anti-LIN28B antibody. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05, **p < 0.01. B Schematic of RNA-pulldown and representative western blots of LIN28B and nucleolin in input and RNA-pulldown samples. CDS coding sequence. C Representative interaction heatmap between TP53 mRNA (5′UTR) and LIN28B protein by catRAPID prediction. D Secondary structure of TP53 mRNA (5′UTR) by RNAfold prediction. E Schematic diagram of truncation mutations of TP53 5′UTR and representative western blots of LIN28B, nucleolin and β-actin in input and RNA-pulldown samples. Exp. = exposure time. *p < 0.05.

Fig 5: LIN28B competes with RPL26 at the TP53 mRNA 5′UTR and inhibits its translation enhancer function.A Representative western blot of LIN28B and RPL26 in A2780 cells with RNA pulldown using control RNA (no biotin) and biotin-labeled TP53 5′UTR sequence. B Representative western blots of RPL26 immunoprecipitation in control (shCtrl) and knockdown (shLIN28B) A2780 cells (left). qPCR of TP53 mRNA after RNA-IP using isotype control IgG or anti-RPL26 antibody in control (shCtrl) and knockdown (shLIN28B) A2780 cells (right). 1, TP53 5′UTR primer #1; 2, TP53 5′UTR primer #2. 18 S served as an internal control. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. C Representative western blots of RPL26 immunoprecipitation in TOV-112D cells transfected with vector control or with LIN28B overexpression (left). qPCR of TP53 mRNA after RNA-IP using isotype control IgG or anti-RPL26 antibody in TOV-112D cells transfected with vector control or with LIN28B overexpression (right). 1, TP53 5′UTR primer #1; 2, TP53 5′UTR primer #2. 18S served as an internal control. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. D Luciferase activity of the TP53 5′UTR reporter with RPL26 and/or LIN28B overexpression in HeLa cells. Representative data of three independent experiments (mean ± s.e.m.). *p < 0.05. E Representative western blots of p53, RPL26, and LIN28B in RPL26 and/or LIN28B-overexpressing HeLa cells. Camp. camptothecin (2 μM, 8 h); endo. endogenous. Listed numbers represent the relative expression of p53.