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.
Our research is aimed at providing the foundation for understanding the molecular mechanisms that maintain the pluripotent state of human ES cells and enhance reprogramming of adult cells. This in turn helps us to design novel strategies to distinguish differentiated from pluripotent stem cells for mass production of cells for therapy, manipulate stem cells to differentiate into specific cells types and enhance reprogramming of patient-specific adult cells for disease modeling and screening of compounds for new drugs. In particular, the generation of disease-specific and genetically diverse stem cell lines aided by our research will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicological testing. This knowledge base will directly support our efforts as well as other Californian researchers to study stem cell biology and design new therapies, and keep California's position as a strong leader in clinical research developments.
Human stem cells maintain a state of self-renewal and pluripotency through a variety of genetic regulatory mechanisms. While significant amounts of work has been focused on understanding the ways in which epigenetic and transcriptional control of gene expression keeps stem cells from spontaneously differentiating to specific lineages, very little has focused on the post-transcriptional control (PTC) of cellular homeostasis. In our proposal, we set out to solve the major problem of which genes are direct targets of microRNAs and an RNA binding protein LIN28, together they define two modes of PTC of expression in pluripotency and reprogramming. In the first year of funding, we have made significant progress in elucidating the regulatory network of RNAs regulated by LIN28. A fraction of our results has been accepted for publication, but the remainder of the project is still underway. We have adapted new advanced genome-wide approaches to studying LIN28's function in the first year of funding, and we expect to reveal unexpected findings in the next year. We have also initiated Aims 2 and Aims 3 of our proposal, which is to define the miRNA targets in human pluripotent stem cells, and to compare these targets with LIN28 targets. In the next two years of funding, we expect to successfully accomplish our proposed aims to reveal the importance of PTC in controlling pluripotency and reprogramming of human stem cells.
Human embryonic stem cells maintain a state of self-renewal and pluripotency through a variety of genetic regulatory mechanisms. While we know a lot about how epigenetic and transcriptional control of gene expression affects cellular homeostasis, few studies have focused on the post-transcriptional control (PTC) of gene regulatory networks. In this proposal, we aim to identify which mRNAs are direct targets of microRNAs and the RNA binding protein LIN28. MicroRNAs and LIN28 are both important in stem cells and misregulation of either can cause cancer. We have adapted new advanced genome-wide approaches to analyze LIN28's function in human stem cells and stable lines over-expressing LIN28 (to mimic cancer situations). The first and second year of funding has enabled us to identify more than 6000 genes that are direct targets of LIN28. A fraction of our results has been published, and the remainder is currently under review. We are currently comparing miRNA targets in
human pluripotent stem cells to LIN28 targets. In the next year of funding, we expect to accomplish our proposed aims to reveal the importance of PTC in controlling pluripotency and reprogramming of human stem cells.
To gain insights into the importance of RNA regulation in embryonic stem cell self-renewal and pluripotency, we have chosen to study the RNA binding protein LIN28, one of the key genes important in stem cell biology as well as a factor in reprogramming somatic cells into induced pluripotent stem cells. In addition, the LIN28 gene is important in cancer (usually highly expressed), diabetes and obesity. Most of its role in biology has been thought to occur through the microRNA let-7. In this award period, we completed our objective to identify other RNA targets of LIN28. In doing so we have performed a genome-wide nucleotide-level resolution map of LIN28 binding sites in messenger RNAs in human embryonic stem cells and somatic cells expressed a tagged version of LIN28. Surprisingly, we find that LIN28 binds to these mRNAs and changes the expression levels of splicing factors (other RNA binding proteins) - which results in alternative splicing changes in a large network of genes. Furthermore LIN28 also regulates itself. Our results suggest novel mechanisms of gene regulation by LIN28 independent of the let-7 microRNA. We have published this in the journal Molecular Cell
- Annu Rev Biochem (2015) The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion. (PubMed: 25784054)
- Mol Cell (2012) LIN28 Binds Messenger RNAs at GGAGA Motifs and Regulates Splicing Factor Abundance. (PubMed: 22959275)
- Nat Neurosci (2012) Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs. (PubMed: 23023293)
- Front Neurosci (2012) Genome-wide approaches to dissect the roles of RNA binding proteins in translational control: implications for neurological diseases. (PubMed: 23060744)
- Nat Struct Mol Biol (2011) LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. (PubMed: 21297634)