Fig. 6: Interplay between Lin28 and YB-1 in cells.

a Left panel: Western blot analysis of whole cell lysate (WCL) and anti-GFP immunoprecipitates (IP) of HEK293 cells expressing GFP or Lin28- GFP with or without RNase treatment. (Upper Gel: only anti-YB-1 antibody. Lower Gel: Anti-GFP and anti-YB-1 antibodies). Right panel: Co-immunoprecipitation of endogenous YB-1 with wild-type Lin28, Lin28-RS or –QS. IP fractions show a smaller amount of YB-1 co-precipitating with the two Lin-28 mutants comparing to wild type Lin28. Three independent experiments are shown. Anti-GFP and anti-YB-1 antibodies were used. b Left panel: Representative images of stress granules in arsenite-treated HeLa cells expressing YB-1-HA and GFP-labeled RBPs as indicated. Zooms in on stress granules show the relative enrichment of YB-1 and GFP-labeled RBPs and the presence of mRNA (in situ hybridization with oligo-d(T) probes). Right panel: Ratio of the enrichment of YB-1 versus indicated RBPs in stress granules for similar expression levels (see Methods). n = 21. **p < 0.01, t-test with two tails versus Lin28. Scale bars: 15 µm and 2 µm (higher magnification, lower panel). c Left panel: Stress granules are detected in arsenite-treated HeLa cells expressing Lin28-GFP using CellProfiler. Anti-YB-1 (Red). The enrichment of YB-1 and Lin28 in stress granules is then measured and plotted to quantify the slope of their relative enrichments. Upper right panel: Scheme representing the consequences of decreasing YB-1 expression on the relative enrichment of YB-1 in stress granules. Lower right panel: Slopes of YB-1 versus wild type or mutant GFP-labeled Lin28 enrichments in stress granules after decreasing or not YB-1 levels are represented. Ratios are given with 95 % confidence bounds. Scale bar: 15 µm. d RT-qPCR analysis of the mRNA content of the anti-GFP immunoprecipitates (IP) in HEK293 cells expressing Lin28-GFP (RS mutant or wild type) with (red) or without (blue) decreasing endogenous YB-1 levels with siRNA. mRNA was extracted from the IP fraction using the standard protocol (see Materials and Methods), then RT-PCR measurements were performed to reveal the presence of mRNAs encoding for genes indicated on right panel. The genes were chosen according to their abundance in HEK cells to avoid imprecise measurements. We noticed that the enrichment of mRNAs in the IP fraction increased significantly when YB-1 levels were decreased for the Lin28-RS mutant compared to wild type Lin28. YB-1 is therefore a competitor for the binding of Lin28-RS to mRNA most probably because of an impaired cooperative association with YB-1 to mRNA.