Uncovering global microRNA roles in development and cancer

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis. Vita.Includes bibliographical references.MicroRNAs (miRNAs) constitute a class of ~22 nucleotide RNAs with broad regulatory roles in gene expression. Dependent largely on the enzyme Dicer for their generation from longer precursor transcripts, mammalian miRNAs direct posttranscriptional repression of mRNAs based on complementarity to sites in their 3' UTRs. To better understand how these regulators impact fundamental processes such as development and cancer, we explored the functional consequences of Dicer loss and subsequent miRNA loss across a range of embryonic and somatic tissues. In embryonic stem (ES) cells, we identified a latent susceptibility to genotoxic stress following deletion of Dicer. Re-expression of the abundant miR-290-295 cluster or knockdown of two novel targets, Caspase 2 and Ei24, could partially restore cell survival after DNA damage, implicating them as important players in a larger stress-responsive ES network under miRNA control. To better understand changes in miRNA and target repertoires at a global scale, we applied a novel evolutionary analysis to the mouse genome designed to speciesspecific innovations. Using this method, we uncovered the genome-wide signature of miRNAs functionally related to the miR-290-295 cluster that we term the Sfmbt2 cluster. In addition to ES cells, placental tissues express these miRNAs at high levels suggesting that mice have co-opted an existing proliferative network to support rapid placental growth. Finally, we evaluated Dicer loss in two transformed somatic cell types, namely sarcoma cells and mesenchymal stem cells. Surprisingly, these cells tolerated Dicer deletion without loss of viability and retained several properties of their Dicer intact counterparts including surface marker expression and tumorigenicity. Comparison of expression data in these cells and ES cells revealed that while many miRNA targets show relatively little change before and after Dicer loss, a subset of genes that differ between embryonic and somatic cells may be controlled in large part by cell type specific miRNAs. In summary, these data shed light on many fundamental aspects of miRNA function in mammalian cells, expanding our understanding of molecular targets as well their downstream cellular roles. As our knowledge about short RNA regulation grows, we are sure to continue uncovering important connections between post-transcriptional regulation and the underlying biology of human development and disease.by Arvind Ravi.Ph.D