RNA polymerase III (RNAPIII) transcribes short, highly structured non-coding RNAs involved in diverse cellular
processes, including translation, transcription regulation, and splicing. The biomedical relevance of RNAPIII
activity in humans is wide-ranging: tRNA, 5S rRNA, vault RNA, 7SL RNA, and small NF90-associated RNA
(snaR) have been shown to be either elevated or depleted in tumors, neurodegenerative brain tissues, viral-
infected B cells, parasite-infected macrophages, and HER2-positive breast cancer cells, respectively. While these
and other examples highlight aberrant activities in distinct health-related contexts, a growing body of evidence
suggests that multiple RNAPIII-transcribed gene subclasses play important roles in the context of heart
development and disease: tRNA and other small RNA levels are altered during early stages of cardiac
differentiation and depleted in response to hypoxia, loss of 7SK RNA is sufficient to induce cardiac hypertrophy,
RMRP RNA is elevated in mouse models of cardiac hypertrophy and in patients with ischemic heart-failure, and
RNAPIII-transcribed Y RNA confers cardioprotection to oxidatively stressed cardiomyocytes. Despite these
findings, little is currently known about the functional role of dynamic non-coding RNA levels within these
contexts, or whether differences in RNA levels are driven by RNAPIII transcription due in part to several unique
challenges related to the sequencing and alignment of short, highly structured and repetitive RNAs. The
proposed study seeks to address these deficiencies by developing new high-throughput genomic methods for
profiling RNAPIII transcription and applying these strategies in the context of stem cell-to-cardiomyocyte
differentiation. In addition, functional experiments will test the role of dynamic RNAPIII patterns within these
contexts, and a genetic-background correction method will be developed to enable future comparisons of
RNAPIII transcription across diverse cellular contexts, accounting for differences in copy number variation
(CNV) in samples of non-identical genomic origin. The proposed study is the next logical step in my progression
to becoming an independently funded investigator with an active and successful research program identifying
the role and underlying regulatory mechanisms of RNAPIII transcription within diverse cellular contexts and
human disease. This project will provide critical training in human stem cell and cardiac biology, CNV detection
methodology, and computational biology and statistics, critical skills necessary to establish my research program
and accomplish my long-term goals as an independent group leader. Together, results of this study promise to
establish improved genomic tools of significant interest to the scientific community, and to yield important
insight on the role and regulation of RNAPIII-transcribed genes during cellular differentiation.
