Examination of mammalian microRNAs by high-throughput sequencing

Small non-coding RNAs play an important role in a wide range of cellular events. MicroRNAs (miRNAs) are an abundant class of small RNAs that post-transcriptionally repress expression of their target genes. Since miRNA targeting is based on its sequence, accurate and comprehensive annotation of miRNA genes is fundamental to understanding miRNA gene regulation. Advances in high-throughput sequencing technology have led to discoveries of novel small RNA genes and identifications of their properties. We describe a method for construction of small-RNA library for Illumina sequencing platform that improves upon previous efforts. Sequencing data from small-RNA libraries constructed using this protocol can be used to profile small RNAs from a broad range of samples. In particular, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. The analysis of the data provide a substantially revised list of confidently identified murine miRNAs, thereby providing a more accurate picture of the general features of mammalian miRNAs and their abundance in the genome. In addition, our results revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan pre-miRNA, cases of consequential 5' heterogeneity, newly identified instances of miRNA editing, and widespread pre-miRNA uridylation reminiscent of Lin28-like miRNA regulation. Thesis Advisor: David P. Bartel Title: Professor of Biology I would like to thank my collaborators who have studied murine miRNAs with me, especially Lori Schoenfeld, Wendy Johnston, Noah Spies, and Vincent Auyeung. I would especially like to thank Graham Ruby for introducing me to computational biology and Daehyun Baek for advices on both scientific and personal endeavors. I would like to thank the members of the Bartel lab for their support and discussion, especially Calvin Jan, Andrew Grimson, Mike Axtell, Anna Drinnenberg, Sue-Jean Hong, Gina Lafkas, Huili Guo, and Vikram Agarwal. I would like to thank my committee members for guiding me through my graduate career: Phil Sharp, Richard Hynes, Chris Burge, and Nelson Lau. I would especially like to thank my advisor Dave Bartel for his guidance and patience throughout the years even as I sometimes struggled to find my way. I would like to thank my classmates in the biology program for the exciting first year in the Pit and friendships throughout the years. I would especially like to thank Leah Okumura, Jen Leslie, Robin Stevens, and Jadyn Damon. I would like to thank the members of the Sidney-Pacific graduate community, in particular the past and present SPEC+ members for being my family away from the family: Swati Mohan, Wendy Iskendarian, Michelle Sanders, Jane Kim, Robert Wang, Matt Eddy, Ben Mares, Alex Lewis, Roger and Dottie Marks, Annette Kim, Roland Tang, and Joshua Tang. I would like to thank my friends who have supported me throughout the years: George Burkhard, Jinhee Chung, Jane Huh, and Jennie Johnson. I would like to thank my family for their support throughout the years, particularly for believing in me and supporting my decision to study abroad. I would especially like to thank my brother HyoSang Chiang for proofreading this thesis. Finally, I would like to thank my fiance Nan Gu for our times together, for more years to come, and for showing me that regardless of the path I choose in life, I do not have to walk it alone.