Drs. Andrew Fire and Craig Mello won this year’s Nobel Prize in Physiology or Medicine for their discovery of RNA interference. Their discoveries revealed fundamental new paradigm in gene regulation and demonstrated that animal genomes consist of not only the transcriptional programs controlled by transcription factors but also the post-transcriptional programs controlled by RNAs. MiRNAs are ~22-nt small regulatory RNAs that are thought to control gene expression at the post-transcriptional level by targeting cognate target mRNAs for either degradation or translational repression. MiRNAs are individually encoded by their own set of genes and represent an integral component of animal genetic programs. MiRNA genes constitute about 1-5% of the predicted genes in worms, mice, and humans, and many miRNAs are conserved from worm to human. Moreover, each miRNA has the potential to regulate as many as 200 target genes, which implies that miRNA-mediated gene regulation may have a broad impact on gene expression and likely represents a fundamental layer of the genetic programs at the post-transcriptional level. Not surprisingly, miRNAs have been shown to play important roles in regulating various cellular, developmental, and disease processes in worm, fly, mouse, and human and several lines of evidence have implicated miRNAs in the developmental regulatory decisions of stem-cell maintenance and differentiation in flies and mice. However, the roles of miRNAs in human embryonic stem cell maintenance and differentiation are still unknown. The proposed research plan will address the fundamental questions regarding the roles of miRNAs and miRNA-mediated posttranscriptional genetic programs in human embryonic stem cells.
Statement of Benefit to California:
To realize the clinical potential human embryonic stem cells, one has to understand the fundamental molecular mechanisms that govern stem cells self-renewal and differentiation. Human embryonic stem cells have a number of important properties: they can propagate under the right culture conditions and they can differentiate into cell types of all human tissues if properly guided. However, until today the molecular processes that regulate these properties are still quite elusive. The proposed research plan will address the fundamental molecular mechanisms that govern stem cells self-renewal and differentiation from a new angel –– the recently discovered post-transcriptional genetic programs controlled by small regulatory RNAs. Thus, these studies are likely to reveal important missing puzzle pieces that may be required for solving the ultimate question and yield clues to harness the power of human embryonic stem cells.
SYNOPSIS: The investigator proposes three specific aims: 1) to establish the role of the miRNA pathway in hES cell maintenance and differentiation; 2) to delineate the function of individual miRNAs in hES cell maintenance and differentiation and 3) to dissect the genetic programs regulated by hES cell miRNAs. miRNA knockdown, lentiviral vector mediated enforced expression, miRNA cloning and quantitation of the levels of individual miRNAs by PCR are proposed. SIGNIFICANCE AND INNOVATION: miRNA is indeed an important mechanism of gene regulation about which relatively little is known. This is an important problem, and the work has the potential to yield significant insights. The PI will perform a comprehensive analysis of miRNAs and miRNA pathways in hESC self renewal and differentiation. The computational and expression cloning methods for identifying miRNA targets appear to be innovative. STRENGTHS: The proposal is from an enthusiastic, productive young investigator who has made a significant contribution to the miRNA field already while working as a post doc and therefore is familiar with the techniques. This investigator also has strong prior experience in analyzing miRNAs in the context of the hematopoietic system. The proposed experimental and computational approaches are comprehensive and likely to yield important insights if suitably developed. WEAKNESSES: An enormous amount of work is proposed. The approach is broad-based and likely to be pursued by many laboratories. This leads to a diffuse and overly ambitious proposal in a field that will have much competition. While the molecular techniques are established, the biological endpoints are rather gross and ill defined. The relevant technical details are lacking, as is an acknowledgement of the potential pitfalls. Specifically, with respect to the first aim the investigator fails to acknowledge the difficulty in identifying effective shRNAs. RNA knockdown or enforced overexpression of one or more of the components of the miRNA pathway may be lethal to cells, or could markedly decrease proliferation so that recovery of such cells may be compromised. Aim 2 is more likely to yield interpretable data, although at this stage it is purely descriptive. With respect to aim 3, it would be helpful if the investigator had given an example of how the proposed approach had identified a miRNA and target of interest. The subsequent analysis of functions of particular miRNAs in hES cell self renewal and/or differentiation would need to be pursued in greater depth to yield substantial insight. It is unlikely that the work can be performed as described in two years. It may have been more appropriate for the PI to narrow the focus, perhaps on aim 2, and more fully develop how the results would be obtained and interpreted. DISCUSSION: The computational and genomic approaches are well combined here, and reviewers like the use of bioinformatics to find miRNA targets, especially since the PI is a young investigator with strong prior experience in this area. However, Aim 1 is not very incisive and could yield little useful information unless the miRNA manipulation gives rise to a specific phenotype. Aims 2 and 3 are strong, but not terribly innovative. Aim 2 could have been developed more fully, and one reviewer noted that the inventiveness in Aim 3 lies in improving the computer target search algorithms and doing the expression cloning. Reviewers commented that eliminating Aim 1 and focusing on Aims 2 and 3 would make this a much stronger proposal.