In addition to the transcription of protein coding genes in the genome, a large amount of transcription encodes RNA molecules that do not generate mRNA. These noncoding RNAs play important roles in the cell that include regulating dosage compensation, controlling genomic imprinting and regulating transcription. However, human cells transcribe thousands of noncoding RNAs and we have only ascribed functions to a small number. One of the main challenges to understanding the functions of noncoding RNAs is that technologies to rapidly identify and characterize noncoding RNAs are lacking. In this proposal, we develop a novel method that makes it possible to identify, in any cell type, all of the noncoding RNAs that interact with chromosomes and at the same time map the sites where those RNAs bind chromatin. Our approach involves directly linking noncoding RNAs to the underlying DNA by generating a covalent chimera between a chromosome bound RNA and DNA. Using next generation sequencing, we can identify the RNAs in the cell that are likely to regulate chromosome structure or function and define their sites of action on the chromosome. In our first Aim we use Drosophila cells to develop this approach, taking advantage of the fact that established chromosomal RNAs, roX1 and roX2, are known to coat the X chromosome to accomplish dosage compensation in the fly. We then broaden this approach in Aim 2 and identify the RNAs that bind chromatin throughout the human genome and develop a new analytical infrastructure to classify and functionally assign these RNAs.
In Aim 3 develop perturbation experiments to test the functions of noncoding RNAs and RNA motifs for their impact on local chromosome accessibility, histone modification state and transcriptional output. We apply a system to redirect noncoding RNAs to new genomic regions to test their functional impact on chromosomes and to regulate different genomic regions through RNA dependent control. By defining the landscape of chromatin associated RNAs in humans and the sites that they regulate in the cell our proposal how these RNAs function as well as the impacts of defects in RNA dependent control that result in cellular dysfunction.

Public Health Relevance

Deregulation of cell type specific gene expression causes developmental defects and drives the progression of human diseases. It is only recently appreciated that a major control mechanism for gene expression is through the actions of non-protein coding RNAs. In this proposal we develop a new method for identifying noncoding RNAs that control gene expression and chromosome function as a route to understanding their functions in human disease.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
1R01HG009909-01
Application #
9427779
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Pazin, Michael J
Project Start
2017-12-01
Project End
2021-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304