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Principal Investigator: Shou-Jiang Gao
Organization: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
Fiscal Year: 2024
Award: $344,531
Funding agency: National Cancer Institute
N6-methyladenosine (m6A) is the most abundant internal modification on poly(A) RNA. Dynamic regulation of
the m6A epitranscriptome is involved in diverse cellular functions. m6A mediates these functions by affecting
mRNA translation, alternative splicing, nuclear export and degradation, and miRNA biogenesis and binding
through three groups of proteins: methyltransferases or “writers”, demethylases or “erasers”, and m6A-binding
proteins or “readers”. m6A is present in the genomes of RNA viruses and regulates the replication of these
viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and
primary effusion lymphoma (PEL) commonly found in AIDS patients. Understanding the mechanism regulating
KSHV latent and lytic replication can not only provide insights into the pathogenesis of KSHV-induced cancers
but also serve as the basis for developing novel therapy. In the current funding period, we have made
significant progresses toward this goal. For this renewal, we have discovered that m6A is abundant on KSHV
transcripts, and that m6A reader protein YTHDF2 acts as an antiviral factor during viral lytic replication. We
demonstrate m6A dynamics during KSHV latent and lytic replications, and in different cell types that support
distinct viral replication programs. Despite these works, the roles of m6A in KSHV infection have just started to
be revealed. The Objective of this application is to systematically map the dynamics of m6A modifications at a
single base resolution and bindings of m6A reader proteins in KSHV epitranscriptome, and determine their
functions in different phases of KSHV life cycle, and KSHV-induced tumorigenesis. The Central Hypothesis is
that KSHV m6A modifications are dynamically regulated, and these modifications mediate different phases of
KSHV life cycle, and hence KSHV-induced tumorigenesis. We will test this hypothesis by mapping m6A marks
in KSHV transcriptome at a single base resolution, and determine the roles of m6A writer and eraser proteins in
different phases of KSHV life cycle and KSHV-induced tumorigenesis (Aim 1); determining the functions of
m6A reader proteins in different phases of KSHV life cycle by gain- and loss-of-function approaches and by
examining their bindings to KSHV transcripts (Aim 2); and examining the functions of KSHV m6A marks in the
context of viral infection using Crispr-Cas9-guided m6A writer and eraser, and by site-specific mutagenesis
(Aim 3). It is our expectations that this project will provide comprehensive mapping and functional delineation
of m6A marks, and m6A writer, eraser and reader proteins in different phases of KSHV life cycle, and KSHV-
induced tumorigenesis. This work is highly significant as it will, for the first time, systematically reveal the
functions of these RNA modifications in KSHV infection, thus providing insights into the mechanism regulating
KSHV life cycle and KSHV-induced pathogenesis. This study will also identify potential prognostic markers and
therapeutic targets. The proposed work is highly innovative as it will use new technologies to map m6A marks
and bindings of m6A reader proteins, as well as directly manipulate m6A marks on key viral transcripts by
Crispr-Cas9-guided m6A writer and eraser, KSHV reverse genetics, and innovative tumor models.
Furthermore, the generated datasets, information and reagents will be valuable to the scientific community.
Terms: <AIDS><Acquired Immune Deficiency><Acquired Immune Deficiency Syndrome><Acquired Immunodeficiency Syndrome><Affect><African><Alternate Splicing><Alternative RNA Splicing><Alternative Splicing><Anti-HIV Positivity><Binding><Binding Proteins><Biogenesis><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancer Induction><Cancers><Cas nuclease technology><Causality><Cell Body><Cell Function><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular Transformation><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Communities><Country><Data Set><EC 2.1.1><Etiology><Funding><Gene Transcription><Genetic Transcription><Genome><Goals><HAART><HHV-8><HHV8><HIV Positive><HIV Positivity><HIV Seroconversion><HIV Seropositivity><HIV antibody positive><HTLV-III Seroconversion><HTLV-III Seropositivity><Herpes infection><Herpesviridae Infections><Herpesviridae disease><Herpesvirus Infections><Highly Active Antiretroviral Therapy><Human><Human Herpesvirus 8><Immune Precipitation><Immunity><Immunoprecipitation><Individual><Intervention><Intervention Strategies><KSHV><Kaposi Sarcoma><Kaposi Sarcoma-Associated Herpes Virus><Kaposi Sarcoma-Associated Herpesvirus><Kaposi sarcoma associated virus><Kaposi sarcoma herpes virus><Kaposi's Sarcoma><Kaposi's sarcoma (KS)-associated herpesvirus><Knock-out><Knockout><Life Cycle><Life Cycle Stages><Ligand Binding Protein><Ligand Binding Protein Gene><Malignant Neoplasms><Malignant Tumor><Maps><Mediating><Messenger RNA><Methyltransferase><Micro RNA><MicroRNAs><Modeling><Modern Man><Modification><Molecular Interaction><Morbidity><Morbidity - disease rate><Multicentric Angiofollicular Lymphoid Hyperplasia><Multicentric Castleman's Disease><Multiple Hemorrhagic Sarcoma><Non-Polyadenylated RNA><Nuclear Export><Nucleotides><Oncogenesis><Origin of Life><Pathogenesis><Patients><Phase><Point Mutation><Poly(A)+ RNA><Polyadenylated RNA><Population><Prevention><Prognostic Marker><Protein Binding><Protein Overexpression><Proteins><RIP seq><RIPseq><RNA><RNA Degradation><RNA Expression><RNA Gene Products><RNA Splicing><RNA Viruses><RNA immunoprecipitation and sequencing><RNA immunoprecipitation sequencing><Reader><Reagent><Regulation><Resolution><Ribonucleic Acid><Role><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Societies><Splicing><Staining method><Stains><Subcellular Process><System><Targeted DNA Modification><Targeted Modification><Testing><Time><Transcript><Transcription><Translations><Tumor Cell><Viral><Viral Diseases><Viral Latency><Virus Diseases><Virus Latency><Virus Replication><Virus-HHV8><Work><base><bases><bound protein><carcinogenesis><causation><cell type><clinical relevance><clinically relevant><crosslink><disease causation><epitranscriptome><expectation><gain of function><global gene expression><global transcription profile><innovate><innovation><innovative><insight><interventional strategy><kaposi's sarcoma herpesvirus><kaposi's sarcoma-associated human herpesvirus><knock-down><knockdown><life course><loss of function><lytic replication><lytic viral replication><lytic virus replication><mRNA><mRNA Translation><malignancy><metaplastic cell transformation><methylase><miRNA><miRNAs><mortality><multicastleman's diseases><neoplasm/cancer><neoplastic cell><new drug treatments><new drugs><new pharmacological therapeutic><new technology><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel technologies><novel therapeutics><novel therapy><overexpress><overexpression><primary effusion lymphoma><prognostic biomarker><programs><resolutions><reverse genetics><social role><therapeutic target><transcriptome><translation><transmethylase><tumor><tumorigenesis><viral infection><viral multiplication><viral replication><virus infection><virus multiplication><virus-induced disease>