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: Direct LIN28B binding and LIN28B-dependent changes in gene expression are uncorrelated(A) Scatterplot showing that total mapped reads per transcript normalized for abundance (normalized eCLIP RPM) is uncorrelated to the fold change in polysome enrichment per transcript, at LIN28 expression level [X4] compared with control HEK293 that do not express LIN28B.(B) The same dataset plotted in (A) but split into gene sets that are bound or not bound by LIN28B and plotted as distributions relative to the fold change polysome enrichment per transcript at LIN28B expression level [X4] compared with control HEK293 that do not express LIN28B.

Fig 4: Characteristics of LIN28B binding on non-miRNAs(A) Changes in read density at any binding site can be plotted as a function of LIN28B expression level, yielding a regression and slope. The slope is correlated to strength of LIN28B binding.(B) Distribution of slopes (µ), encompassing 11,433 target sites.(C) Frequency of known LIN28 motifs, ‘‘UGAU,’’ in eCLIP-identified binding sites.(D) Frequency of known LIN28 motifs, ‘‘GNNG,’’ in CLIP-identified LIN28 binding sites.(E) Boxplots showing the distribution of GC, GU, AU, and GG pairs found in RNA structures formed in LIN28 binding sites.(F) Cumulative distribution functions showing the minimum free energy of the ensemble for all higher-affinity binding sites (m < 0, solid red), the 100 highest-affinity binding sites (dashed red), all lower-affinity sites (m > 0, solid green), and the 100 lowest affinity binding sites (dashed green) compared with randomly chosen sequences outside of eCLIP-identified binding sites (gray).(G) Receiver-operator characteristic for two random forest classifiers that were developed to predict the affinity of a LIN28 binding site (blue) or the location of a LIN28 binding site (yellow). Predictions against data with shuffled identifiers were used as a control (dashed).(H) Boxplots showing the accuracy of random forest classifier predictions compared with performance on a dataset with shuffled identifiers.

Fig 5: DDX3X-L556S patient-derived iPSCs escape X inactivation(A) Immunofluorescence for LIN28 (red), SSEA4 (green), and DAPI (blue) in WT-iPSC and DDX3X-L556S iPSCs. Scale bar, 20 µm.(B) Microarray comparative genomic hybridization (Array-CGH) analysis for genome-wide chromosomal imbalances shows all iPSC clones derived from the patient with L556S mutation used in this study present no chromosome copy number alteration.(C and D) (C) RT-qPCR analysis of iPSCs for NANOG and (D) OCT3/4 expression. Error bars ± SEM. n.s: not statistically significant, Student's t t test.(E) Immunofluorescence of WT-iPSC and DDX3X-L556S iPSCs for DDX3X (green), OCT4 (red). Scale bar, 20 µm.(F) RT-qPCR analysis of iPSCs culture of WT and DDX3X-L556S for DDX3X expression. Error bars ± SEM, Student's t test.(G) Sequencing chromatograms demonstrating that the mutated L556S DDX3X allele is transcribed and escapes X inactivation in iPSCs and blood cells DDX3X-L556S patient (black arrows), compared with WT blood cell sample.