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. 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).