Gene expression is mediated by DNA- and RNA-binding proteins in every organism. Regulation of gene expression is commonly mediated by protein modifications of these nucleic acid binding proteins. This proposal focuses on an under-explored but therapeutically important protein modification called poly(ADP- ribose) (PAR). PAR has been well known for its roles in DNA repair and transcription in the nucleus. Recently, we discovered that PAR also modifies several post-transcriptional mRNA gene regulators in the cytoplasm. Our data are consistent with recent proteomics studies showing that poly(ADP-ribosyl)ated (PARylated) proteomes are enriched with RNA-binding proteins, suggesting that PAR plays a much broader regulatory role in RNA metabolism than previously appreciated. In this proposal, we will focus on how PAR regulates microRNA functions. MicroRNAs are a class of ~22 nucleotide non-coding RNAs that regulate many fundamental cellular processes, including stress responses. Although much has been characterized about microRNA biogenesis, little is known about how microRNA activities are regulated. Key data: Recent data including ours indicate that microRNA activities are inhibited by the PARylation of the core microRNA-binding protein Argonaute (AGO). Such inhibition is regulated by PAR polymerase 13 (PARP- 13) where its overexpression reduces microRNA activities. Intriguingly, PARP-13 is catalytically inactive; therefore, other catalytically active PARP(s) must be involved. Such a PARylation mechanism involving more than one PARP represents a new paradigm. In this proposal, we will investigate how PARP-13 interacts with a cytoplasmic, catalytically active PARP to PARylate AGO (Aim 1), determine how PAR polymers on AGO reduce microRNA activities (Aim 2) and identify which domains of AGO are modified by PAR (Aim 3). We will use a novel mass spectrometry technique to identify AGO PARylation sites. Until now, the identification of PARylation sites has been a challenge for the field and thus this study allows the first systematic analysis of the functional roles of individual sites of a protein substrate. O note, PARP-13 is also known as zinc antiviral protein (ZAP) - a host factor that inhibits the replication of Sindbis virus, Ebola virus, Hepatitis B virus and HIV upon overexpression. Therefore, AGO PARylation-mediated inhibition of microRNA activities may be involved in host antiviral responses, which we will explore in the context of Sindbis virus infection (Aim 3c) in collaboration with Dr. Diane Griffin at Johns Hopkins. The Team: To ensure success, this project is performed with two key collaborators (both of whom we are requesting for one funding module): AGO biochemistry expert Dr. Leemor Joshua-Tor (Cold Spring Harbor Laboratory) and proteomics expert Dr. Shao-En Ong (University of Washington). Other consultants include Drs. Phillip Sharp (MIT) and Carl Novina (Harvard) on microRNA biology, Drs. Ted Dawson (John Hopkins) and Paul Chang (MIT) on PAR biology, and Dr. Pierre Coulombe (Johns Hopkins) on biochemistry.

Public Health Relevance

This proposal examines the interplay between two therapeutically important molecules - microRNA and poly(ADP-ribose). As microRNA activities are regulated by the level of poly(ADP-ribose) via a cellular protein that has anti-viral functions against Ebola virus, Hepatitis B virus and HIV, our study may open up new antiviral therapeutic opportunities.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Bender, Michael T
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Johns Hopkins University
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McPherson, Robert Lyle; Ong, Shao-En; Leung, Anthony K L (2018) Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry. Methods Mol Biol 1813:271-283
Abraham, Rachy; Hauer, Debra; McPherson, Robert Lyle et al. (2018) ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication. Proc Natl Acad Sci U S A 115:E10457-E10466
Abraham, Rachy; McPherson, Robert Lyle; Sreekumar, Easwaran et al. (2018) Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation. Methods Mol Biol 1813:297-316
Leung, Anthony K L (2017) PARPs. Curr Biol 27:R1256-R1258
Leung, Anthony K L (2017) SERious Surprises for ADP-Ribosylation Specificity: HPF1 Switches PARP1 Specificity to Ser Residues. Mol Cell 65:777-778
Fischer, Joseph W; Leung, Anthony K L (2017) CircRNAs: a regulator of cellular stress. Crit Rev Biochem Mol Biol 52:220-233
Vivelo, Christina A; Wat, Ricky; Agrawal, Charul et al. (2017) ADPriboDB: The database of ADP-ribosylated proteins. Nucleic Acids Res 45:D204-D209
McPherson, Robert Lyle; Abraham, Rachy; Sreekumar, Easwaran et al. (2017) ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence. Proc Natl Acad Sci U S A 114:1666-1671
Daniels, Casey M; Ong, Shao-En; Leung, Anthony K L (2017) ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method. Methods Mol Biol 1608:79-93
McPherson, Robert Lyle; Leung, Anthony K L (2016) ADPr-ChAP: Mapping ADP-Ribosylation onto the Genome. Mol Cell 61:327-328

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