This competitive revision is being submitted for Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availablity of Recovery Act Funds for Competitive Revision Applications. Background. The ability of short interfering RNAs (siRNAs) to recognize mRNA is widely appreciated. siRNAs are entering Phase 1 trials, but their ultimate potential to impact human health is unclear. The overall goal of the parent proposal GM 77253 was to investigate the action of duplex RNAs that are complementary to gene promoters. GM 77253 had four Aims: i. Evaluate involvement of argonaute proteins in RNA-mediated gene activation ii. Characterize the mechanism of transcriptional silencing by agRNAs iii. Define rules for targeting gene promoters iv. Identify endogenous miRNAs that inhibit transcription Rationale for a significant expansion of research scope. Recent transcriptome studies have revealed many noncoding transcripts overlapping the 3'terminus of genes. The function of these transcripts is unknown and the potential for regulation downstream from the 3'-UTR has attracted little attention. While pursuing Aim ii, we observed that promoter-targeted RNAs were binding to a noncoding transcript that overlaps the 5'terminus of our target gene progesterone receptor. We hypothesized that RNAs that target sequences past the 3'-terminus of genes might also be able to bind noncoding transcripts and modulate gene expression. We characterized transcription at the progesterone receptor (PR) locus and identified noncoding transcripts that overlap the 3'end of the gene. Small RNAs complementary to a noncoding transcript inhibit PR transcription in T47D cells (a cell line with high PR expression) and activate PR transcription in MCF7 cells (a cell line with low PR expression). The RNAs recruit argonaute to the 3'- noncoding transcript, alter levels of RNA polymerase, and modulate transcription of PR pre-mRNA and mature mRNA. RNAs complementary to sequences beyond the 3'mRNA terminus of BRCA1 also inhibit gene expression. Our results extend the potential for RNA-mediated gene regulation to regions downstream from the 3'end of genes. We propose to characterize the mechanism of action of 3'-targeted RNAs to understand how they can modulate transcription even though they are not complementary to mRNA and target sequences that are far distant from gene promoters.

Public Health Relevance

Regulation of gene expression is fundamental to biological processes. Our research investigates regions of chromosomal DNA that lie beyond the termini of genes. These regions have received little attention and we will investigate whether gene regulation can extend to these regions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM077253-03S1
Application #
7833533
Study Section
Special Emphasis Panel (ZRG1-GGG-B (95))
Program Officer
Tompkins, Laurie
Project Start
2009-09-30
Project End
2011-07-31
Budget Start
2009-09-30
Budget End
2011-07-31
Support Year
3
Fiscal Year
2009
Total Cost
$341,714
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Chu, Yongjun; Kalantari, Roya; Dodd, David W et al. (2012) Transcriptional silencing by hairpin RNAs complementary to a gene promoter. Nucleic Acid Ther 22:147-51
Gagnon, Keith T; Corey, David R (2012) Argonaute and the nuclear RNAs: new pathways for RNA-mediated control of gene expression. Nucleic Acid Ther 22:3-16
Younger, Scott T; Corey, David R (2011) Transcriptional regulation by miRNA mimics that target sequences downstream of gene termini. Mol Biosyst 7:2383-8
Schwartz, Jacob C; Corey, David R (2011) Practical considerations for analyzing antigene RNAs (agRNAs): RNA immunoprecipitation of argonaute protein. Methods Mol Biol 764:301-15
Yu, Dongbo; Sakurai, Fuminori; Corey, David R (2011) Clonal Rett Syndrome cell lines to test compounds for activation of wild-type MeCP2 expression. Bioorg Med Chem Lett 21:5202-5
Matsui, Masayuki; Sakurai, Fuminori; Elbashir, Sayda et al. (2010) Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter. Chem Biol 17:1344-55
Janowski, Bethany A; Corey, David R (2010) Minireview: Switching on progesterone receptor expression with duplex RNA. Mol Endocrinol 24:2243-52
Yue, Xuan; Schwartz, Jacob C; Chu, Yongjun et al. (2010) Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini. Nat Chem Biol 6:621-9
Watts, Jonathan K; Corey, David R (2010) Clinical status of duplex RNA. Bioorg Med Chem Lett 20:3203-7
Younger, Scott T; Corey, David R (2009) The puzzle of RNAs that target gene promoters. Chembiochem 10:1135-9

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