A critical control point for regulating gene expression in eukaryotic cells is during mRNA transcription by RNA polymerase II (Pol II). Unexpectedly, non-coding RNA molecules (ncRNAs) have been found to regulate mRNA transcription. Mouse B2 RNA and human Alu RNA are two such ncRNAs;they function as repressors of mRNA transcription by binding directly to Pol II in response to heat shock, a widely used model system for studying the cellular stress response. The long term goal of this research is to understand how mammalian ncRNAs directly control Pol II transcription in biologically important and medically relevant experimental systems. These studies will contribute to discerning how transcription is regulated during cellular stress, which is critical for understanding abnormalities in gene expression associated with diseases and deleterious environmental states. Moreover, our studies will move the fields of transcriptional regulation and functional ncRNAs in new directions. The proposal has three Specific Aims. 1) Determine how B2 RNA controls genome-wide Pol II occupancy and the molecular characteristics of paused complexes in response to heat shock using a combination of genomic assays (e.g. ChIP-seq, RNA-seq) and more targeted assays (e.g. ChIP, permanganate treatment). 2) Identify and study factors that control the activities of B2 RNA and Alu RNA using in vitro protein-RNA interaction assays, cellular knockdown of factors, co-immunoprecipitations, and ChIP. 3) Understand the mechanism and functions of the mammalian Pol II RdRP activity using a combination of RdRP assays, RNA secondary structure analysis, protein-RNA interaction assays, in vitro transcription experiments, and targeted RNA-seq. Together this work will contribute a new understanding of how ncRNAs can globally regulate a critical biological process. Moreover, by using as a model system the widespread, yet transient, transcriptional repression that occurs in response to heat shock, we are uniquely poised to uncover new aspects of transcriptional control of macromolecular complex assembly on promoters and post-initiation events. In addition, we will identify and characterize a global regulator of transcriptional activation in response to heat shock that functions by derepressing the activities of B2 and Alu RNAs. Lastly, our experiments will be the first to characterize mammalian Pol II as an RdRP that acts on cellular RNA templates.

Public Health Relevance

Properly controlling gene expression is essential to sustaining life and avoiding many diseases and cancers;mRNA transcription by RNA polymerase II is central to this process. The proposed studies will define a new means by which programs of mRNA transcription are controlled during cellular stress. Acquiring this knowledge is critical for understanding abnormalities in gene expression associated with diseases and deleterious environmental states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068414-11
Application #
8521313
Study Section
Special Emphasis Panel (ZRG1-GGG-N (03))
Program Officer
Sledjeski, Darren D
Project Start
2003-09-19
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
11
Fiscal Year
2013
Total Cost
$304,241
Indirect Cost
$99,178
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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Wagner, Stacey D; Yakovchuk, Petro; Gilman, Benjamin et al. (2013) RNA polymerase II acts as an RNA-dependent RNA polymerase to extend and destabilize a non-coding RNA. EMBO J 32:781-90
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Kugel, Jennifer F; Goodrich, James A (2012) Non-coding RNAs: key regulators of mammalian transcription. Trends Biochem Sci 37:144-51
Tarallo, Valeria; Hirano, Yoshio; Gelfand, Bradley D et al. (2012) DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell 149:847-59
Kaneko, Hiroki; Dridi, Sami; Tarallo, Valeria et al. (2011) DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature 471:325-30
Goodrich, James A; Kugel, Jennifer F (2010) Dampening DNA binding: a common mechanism of transcriptional repression for both ncRNAs and protein domains. RNA Biol 7:305-9
Wagner, Stacey D; Kugel, Jennifer F; Goodrich, James A (2010) TFIIF facilitates dissociation of RNA polymerase II from noncoding RNAs that lack a repression domain. Mol Cell Biol 30:91-7

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