RNA is perhaps the most versatile molecule in the cell due to its significant contribution to nearly every cellular process. But we still don?t really understand many aspects of how RNA features, such as nucleotide modifications and secondary and tertiary structure, work in concert to regulate its many functionalities. The central dogma of biology tells us that DNA is transcribed to RNA, which then is translated to protein. We already know that DNA has a complex web of structural organization and covalent modification which provides transcriptional control. But modifications, splicing, and structure of RNA involve an even more complex interplay to regulate protein production. Yet without appropriate tools, we cannot explore this world. Here we propose to leverage the newly developed direct RNA nanopore sequencing platform to directly interrogate RNA, enabling us to examine modifications to RNA and their interplay with the dynamics of splicing and RNA structure. Nanopore sequencing directly probes the chemical structure of the molecule in the pore, allowing us to measure endogenous RNA modifications; but we first need to understand the raw electrical signal data produced, through the generation of training sets. To explore the dynamics of RNA production and splicing, we will utilize this toolbox to examine non-canonical nucleotides, e.g. 4sU, that we inject as a metabolic label for nascent RNA. For structural probing, we will adapt conventional techniques which label unpaired RNA nucleotides to allow high-throughput examination of complex RNA structure. These tools and analysis pipelines will grant new insights into mechanisms of transcriptional regulation, as well as their implications for human disease.

Public Health Relevance

The central dogma of biology tells us that DNA is transcribed to RNA, which then encodes protein. But there are complicated regulatory layers which serve to control this biology, layers which are poorly understood and often go awry in disease. We are developing a new toolset which will, for the first time, allow us to examine RNA directly to probe its structure and dynamics, granting new insights into its biology.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
1R01HG010538-01
Application #
9733580
Study Section
Special Emphasis Panel (ZHG1)
Program Officer
Smith, Michael
Project Start
2019-04-22
Project End
2023-01-31
Budget Start
2019-04-22
Budget End
2020-01-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biomedical Engineering
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205