The discovery of reversible N6-methyladenosine (m6A) modification of RNA has fundamentally altered the way we think of the central dogma of molecular biology. M6A is added post-transcriptionally to RNA, where it has been implicated in such diverse processes as RNA splicing, nuclear export, stability, and translation. The mechanisms in which post-transcriptional modifications of RNA controls RNA fate has opened up a new field of biology dubbed ?Epitranscriptomics?. Since small DNA viruses that replicate in the nucleus have to employ cellular machinery to transcribe and translate their gene products, viruses have developed ways to harness cellular RNA processing pathways. Virus infections thus provide elegant biological models to decipher how RNA transcription and its chemical modifications can be regulated and exploited to direct the host cell machinery towards production of viral progeny. Shortly after the discovery of m6A on human RNA in 1974, it was demonstrated that adenovirus RNAs are also marked by m6A. However, the effect of m6A modifications on Adenovirus has never been deciphered and in the intervening 40 years no progress has been made in understanding how epitranscriptomic modifications impact the life cycle of DNA viruses. Adenovirus has served as an excellent model for illuminating fundamental cellular processes such as RNA capping, splicing, and polyadenylation, and I propose to use Adenovirus as a model pathogen to discover novel m6A-regulating factors of mRNA transcripts in human cells. My preliminary data map the site specific locations of m6A-modifications within Adenoviral RNA transcripts. Furthermore, we show that knockdown of the human m6A methyltransferase complex negatively affects Adenoviral late protein expression and infectious virus production. At the same time, the virus recruits members of the host m6A pathway to sites of viral replication.
The specific aims of this proposal are to 1) determine host factors that methylate and bind methylated adenoviral RNA transcripts, 2) determine the function of m6A in the life cycle of adenovirus, and 3) determine the extent that host RNA methylation is altered during viral infection.
In Aim 1 I will investigate the human methyltransferase METTL3 as the possible source of Adenoviral RNA methylation.
In Aim 2 I will explore possible roles for m6A methylation in the adenoviral life cycle such as RNA splicing and export from the nucleus.
In Aim 3 I will define the changing methylation status of host RNA in response to Adenovirus infection. Together, these experiments will provide insight into how DNA viruses interact with and control host RNA biogenesis machinery. The completion of these aims, and the training provided within, will provide the foundation for future investigation on my path to being an independent investigator.

Public Health Relevance

N6-methyladenosine (m6A) is a chemical modification of RNA that has recently been discovered to play diverse biological roles in RNA splicing, nuclear export, stability, and translation. After the discovery of m6A over forty years ago it was shown that RNAs from DNA viruses such as Adenovirus are marked by m6A, however no role has been defined for m6A in the infectious life cycle of DNA viruses. In this proposal, I aim to elucidate the role of m6A in the adenoviral life cycle and use adenovirus as a model pathogen to discover novel m6A-regulating factors of mRNA transcripts in human cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32AI138432-01
Application #
9540335
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Natarajan, Ramya
Project Start
2019-02-01
Project End
2021-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104