Roles of Long Non-Coding RNAs in Human Embryonic Stem Cell Pluripotency and Neural Differentiation

Abstract

Long non-coding RNAs (lncRNAs) are a recently discovered class of transcripts encoded within the human genome. LncRNAs have been proposed to be key regulators of biological processes, including stem cell pluripotency and neurogenesis. However, at present very little functional characterization of lncRNAs involved in differentiation has been carried out in human cells. In this thesis, functional characterization of lncRNAs in human development is addressed using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. Human ESCs were robustly and efficiently differentiated into neurons, and expression of lncRNAs was profiled using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified, and shown to physically interact with SOX2, and PRC2 complex component, SUZ12. Using a similar approach, we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis, and immunoprecipitation studies revealed physical association with REST and SUZ12.

In particular, a neuronal lncRNA, RMST, was found to be essential for neurogenesis. Knockdown of RMST in human neural stem cells prevented neurogenesis. RNA pulldown and RNA immunoprecipitation indicated that RMST physically associated with the RNA-binding protein hnRNPA2B1 and the transcription factor SOX2. Perturbation studies, followed by genome-wide transcriptional profiling indicated that RMST and SOX2 co-regulate a large pool of targets. Interestingly, knockdown of RMST resulted in reduced SOX2 occupancy at its target gene promoters, suggesting that RMST may alter SOX2 binding to chromatin during neurogenesis. Together, this study represents important evidence for an indispensable role of lncRNAs in human brain development.