Non-microRNA Binding Competitively Inhibits LIN28 Regulation

RNA binding protein (RBP) expression is finite. For RBPs that are vastly outnumbered by their potential target sites, a simple competition for binding can set the magnitude of post-transcriptional control. Here we show that LIN28, best known for its direct regulation of let-7 miRNA biogenesis, is also indirectly regulated by its widespread binding of non-miRNA transcripts. Approximately 99% of LIN28 binding sites are found on non-miRNA transcripts, like protein coding and ribosomal RNAs. These sites are bound specifically and strongly, but they do not appear to mediate direct post-transcriptional regulation. Instead, non-miRNA sites act to sequester LIN28 protein and effectively change its functional availability, thus impeding the regulation of let-7 in cells. Together, these data show that the binding properties of the transcriptome broadly influence the ability of an RBP to mediate changes in RNA metabolism and gene expression. Overall design: Examination of LIN28B binding using enhanced CLIP via 2 biological replicates of RNA fragments derived from LIN28B immunoprecipitation and 2 control input replicates for 4 LIN28B expression conditions in HEK293A (LIN28B_X1, LIN28B_X2, LIN28B_X3, LIN28B_X4). Examination of LIN28B binding using enhanced CLIP via 2 biological replicates of RNA fragments derived from Lin28A immunoprecipitation and 2 control input replicates in mouse ESCs with (HPK_mESCs) and without (V6_5_mESCs) sponge expression. Examination of changes in transcriptome-wide ribosome occupancy in mouse ESCs via total RNA (2 replicates, T) and ribosome-bound RNA (2 replicates, P) for several experimental conditions: (dd-Cit = double Dicer knockout ESCs expressing mock Citrine), (dd-HpK = double Dicer knockout ESCs expressing LIN28 sponge), (V6_5_mock = V6.5 ESCs expressing mock Citrine), (Hpk_mock = V6.5 ESCs expressing LIN28 sponge).