RNA Binding Protein-mediated Post-transcriptional Networks Regulating HPSC Pluripotency

Human embryonic stem cells (hESC) have the remarkable capacity to replicate indefinitely and differentiate into virtually any cell type in the human body. Maintaining this pluripotent cell state requires the precise control of hundreds, if not thousands of proteins in the cells, a process known as gene regulation. Recently it has been shown that adult human cells can be induced to revert back to earlier stages of development and exhibit properties similar to hESCs. The exact method for "reprogramming" is still being optimized but currently requires inserting multiple genes into adult cells and then exposing them to the appropriate environment suitable for hESC growth, to produce these "induced pluripotent stem (iPS) cells". Generation of patient-specific iPS cells will be of tremendous benefit to disease-related biomedical research and therapy. It is of interest that many of these genes are hESC enriched or specific to pluripotent stem cells, thus understanding the regulation of genes important for pluripotency is of strong benefit to reprogramming as well.

Genes are regulated at many different levels, beginning with the production of RNAs in the nucleus (transcription), and ending with the generation of proteins from processed RNAs in the cytoplasm (translation). While much is known about the transcriptional control of gene expression involved in maintain the pluripotency of stem cells, relatively little is known about what happens to the RNAs after transcription (post-transcriptional control, or PTC), before translation. RNA binding proteins (RBPs) associate with RNAs during this intermediate stage, several of which bind directly to RNAs (targets), while others interact indirectly with RNAs via small non-coding RNAs called microRNAs to change the expression of target RNAs.

The goal of the proposed research is to produce a comprehensive map of RNAs that are targeted by RBPs important for pluripotency in stem cells, as well as uncover how these RBPs regulate their target RNAs. We will use a modification of a high throughput biochemical strategy to identify the precise location on RNAs that are in contact with carefully chosen RBPs. We will isolate and sequence millions short nucleotides representing stretches of these RNAs and map them to the human genome, together representing the complete post-transcriptional controlled regions of pluripotent stem cells. Completion of the proposed research is expected to improve our understanding of the gene regulatory mechanisms in human pluripotent stem cells, which in turn will facilitate the development of new strategies for stem cell based therapeutics and enhance reprogramming of patient-specific adult cells.