UC San Diego

The field of stem cell biology is moving forward at an unprecedented rate in part due to the discovery that adult somatic cells can be reprogrammed to a pluripotent stem cell like state. The factors first used in reprogramming were transcription factors such as OCT4, SOX2 and NANOG, and the RNA-binding protein LIN28. Like transcription factors, RNA-binding proteins (RBPs) control vast networks of gene targets to direct pathways in the cell; however, for RBPs this is accomplished through post-transcriptional binding to RNA transcripts. Only recently has it been possible to survey the transcripome-wide RNA binding interactions of a protein, through isolation of endogenous RBP-RNA complexes paired with high-throughput sequencing technologies. Using cross-linking followed by immunoprecipitation of protein-RNA complexes and sequencing of isolated transcripts (CLIP-seq) we have identified LIN28 binding sites throughout the human transcriptome. The resolution of our data enabled us to define characteristic LIN28 mRNA interactions at GGAGA rich motifs within unpaired regions of hairpin loops. This binding pattern mimics interactions described for LIN28 binding within let-7 family microRNA precursors. The ability to consider LIN28 targets on a global scale enabled the identification of RNA processing factors, in particular splicing factors, as prevalent functions encoded by LIN28 bound RNAs. This information helped to accurately predict which of the thousands of LIN28 targets would be functionally regulated. We found evidence that LIN28 increases the protein production of splicing factors resulting in massive rearrangement of RNA transcripts through downstream splicing changes. Subsequent transcriptome-wide studies of LIN28 have confirmed these findings despite differences in the pool of direct targets defined by individual reports. Taken together, we understand that LIN28 can bind to a wide network of transcripts, influencing development through these direct RNA interactions and via downstream effects. Combinatorial approaches in the study of LIN28 using changes in RNA- levels, protein production, strength of CLIP-seq binding, and ontological classification of gene targets have extracted meaningful information about mechanisms of LIN28 regulation. We expect that application of similar methods will enable studies of additional RBPs. For example, in the study of other stem cell enriched proteins like the IGFII-mRNA binding proteins (IG2BP or IMP). Furthermore, the overlap of other regulatory networks hold promise of highlighting novel hubs of regulation that may be exploited in reprogramming or directed differentiation. The next step is to use these connections to explain how genetic changes within an individual can affect RBP function and result in disease. We can apply in vitro modeling of development using directed differentiation to iteratively test how the connection of LIN28 to its target transcripts impacts its role in development and disease