Cell viability requires maintenance of genome integrity, which is continuously threatened by intracellular and external sources of DNA damage. One prevalent source of DNA damage is UV light, which generates cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone (6-4 PP) adducts. The nucleotide excision repair (NER) pathway is mainly responsible for the removal and repair of these lesions. Two different branches of the NER pathway contribute to repair: transcription coupled repair (TCR) recognizes CPDs at transcriptionally active loci, while global genomic repair (GGR) marks lesions in other parts of the genome. Trans-lesion synthesis (TLS) operates in parallel to bypass any unrepaired lesions in S phase. Mutations in many of these components cause photosensitivity diseases, notably Xeroderma Pigmentosa and Cockayne Syndrome. Recently, we have identified methylation of RNA at the 6 position of adenosines (6mA) as an exciting new contributor to DNA repair. 6mA RNA is rapidly and transiently induced at damage sites following UV exposure, and we have identified the RNA methyltransferase METTL3 as being responsible for this modification. Our preliminary results suggest that METTL3 may participate in the TCR branch of the NER pathway, as repair of transcribed regions is specifically compromised in METTL3 knock-out (KO) cells. Accordingly, loss of 6mA RNA by METTL3 KO decreased cell survival after UV exposure, which was rescued by wild-type, but not catalytically-inactive, METTL3. The goals of this work are to determine the role and mechanism of action of 6mA in DNA repair. We will determine whether 6mA RNA functions to repair transcribed and/ or non-transcribed regions genome-wide, and identify in which pathways/ steps of NER it participates. We will also explore its role in TLS and repair of other types of DNA damage to determine how broadly 6mA RNA operates in the DNA damage response (DDR). Secondly, we will investigate the hypotheses that 6mA marks transcripts arising from damaged DNA templates for degradation, and/ or that 6mA influences alternative splicing or translation of modified transcripts, particularly of those encoding proteins important for DNA repair or cell survival. Finally, we will identify and characterize reader proteins that recognize 6mA RNA, in order to investigate the hypothesis that these readers play a mechanistic role in linking 6mA RNA to the DDR. Our discovery of an RNA modification mediating DNA repair has uncovered a new and exciting facet to the DDR and to our knowledge of how cells maintain genome integrity. The research proposed in this application will provide important new insight into how 6mA RNA regulates the DDR, and our findings will be important for understanding and treating photosensitivity diseases.

Public Health Relevance

Photosensitivity diseases such as Xeroderma Pigmentosa and Cockayne Syndrome arise from mutations in genes that repair UV-damaged DNA. Although many proteins participating in DNA repair have been described, we have found a new type of molecule-- RNA methylated at the 6 position of adenosines-- that also aids in DNA repair. Our goals are to understand how methylated RNA promotes repair of UV damage and ensures cell survival, which will further our understanding of photosensitivity diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA118487-13
Application #
9321060
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Okano, Paul
Project Start
2006-07-13
Project End
2018-06-30
Budget Start
2017-07-01
Budget End
2018-06-30
Support Year
13
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Boston Children's Hospital
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Sheng, Wanqiang; LaFleur, Martin W; Nguyen, Thao H et al. (2018) LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade. Cell 174:549-563.e19
Li, Haojie; Liefke, Robert; Jiang, Junyi et al. (2017) Polycomb-like proteins link the PRC2 complex to CpG islands. Nature 549:287-291
Xiang, Yang; Laurent, Benoit; Hsu, Chih-Hung et al. (2017) RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543:573-576
Jambhekar, Ashwini; Anastas, Jamie N; Shi, Yang (2017) Histone Lysine Demethylase Inhibitors. Cold Spring Harb Perspect Med 7:
Murn, Jernej; Shi, Yang (2017) The winding path of protein methylation research: milestones and new frontiers. Nat Rev Mol Cell Biol 18:517-527
Laurent, B; Shi, Y (2016) Expression, Purification, and Biochemical Analysis of the LSD1/KDM1A Histone Demethylase. Methods Enzymol 573:241-59
Shen, Hongjie; Xu, Wenqi; Guo, Rui et al. (2016) Suppression of Enhancer Overactivation by a RACK7-Histone Demethylase Complex. Cell 165:331-42
Liefke, Robert; Karwacki-Neisius, Violetta; Shi, Yang (2016) EPOP Interacts with Elongin BC and USP7 to Modulate the Chromatin Landscape. Mol Cell 64:659-672
Ma, Chun; Karwacki-Neisius, Violetta; Tang, Haoran et al. (2016) Nono, a Bivalent Domain Factor, Regulates Erk Signaling and Mouse Embryonic Stem Cell Pluripotency. Cell Rep 17:997-1007
Greer, Eric Lieberman; Blanco, Mario Andres; Gu, Lei et al. (2015) DNA Methylation on N6-Adenine in C. elegans. Cell 161:868-78

Showing the most recent 10 out of 38 publications