Background: Class switch recombination and somatic hypermutation are two B lymphocyte specific processes that mediate antibody diversification. Activation Induced Cytidine Deaminase (AID) is essential for initiating both of these processes by deaminating cytidine residues in immunoglobulin (Ig) loci DNA. Despite its specific and indispensible function in the Ig loci, AID has been demonstrated to also deaminate non-Ig locus genes, catalyzing various oncogenic translocations that manifest in tumorogenesis. Recent work has indicated that AID's DNA deamination activity requires its association with transcriptionally stalled RNA polymerase II and the RNA exosome complex. RNA exosome is a cellular non-coding RNA processing and/or degradation macromolecular complex. It is postulated that RNA Exosome's activity is mediated by specific cofactors and sequence characteristics of the target RNA. How RNA exosome's RNA processing activity facilitates AID mediated DNA deamination is a question we seek to address in this application. Objective/Hypothesis: In his proposal, we will determine how RNA exosome activity on transcripts generated in the IgH locus and the rest of the B cell genome generates single-stranded DNA structures following stalling of RNA polII complex and depletion of nucleosomes. Single-strand DNA structures are suitable AID substrates.
Specific aims :
Aim 1 : Are the DIS3 and Exosc10 RNase subunits of RNA exosome complex important for AID activity?;
AIM 2 : How does RNA exosome substrate antisense RNA (xTSS-RNA and asRNA) promote AID targeting in the B cell genome? AIM 3: To evaluate the role of RNA exosome cofactor Mtr4 (and Senataxin) in the mutagenesis of both strands of DNA in the IgH locus and other regions of the B cell genome. Study Design: Using mouse models that are deficient in RNA exosome RNA degradation activity, we will identify regions in germinal center derived B cell genome that express RNA exosome substrate non-coding RNAs and are also mutated by AID. We will evaluate the mechanism of formation of single-strand DNA structures following localized chromatin remodeling at these identified AID target DNA sequences. We have also generated a mouse model in which the RNA exosome subunit, Exosc3, has been tandem-tagged for high affinity RNA exosome complex purification. Using B cells from these mice, we have purifed RNA exosome complex co-factors and identify them by LC-MS/MS. We will evaluate the role of RNA exosome co-factor(s) in stimulating AID/RNA exosome complex function on both strands of transcribed DNA substrate. Disease Relevance: AID initiates various malignancies in B cells due to its aberrant DNA mutagenesis activity. Proposed studies leads to a better understanding of the mechanisms initiating AID dependent oncogenesis in B lymphocytes (specially in context of DLBCL and Multiple Myeloma), as well as, have direct implications in understanding B lymphocyte based immunodeficiency syndromes like Hyper-IgM syndrome 2.

Public Health Relevance

The correct topological organization of the B-lymphocyte genome during development and antibody gene diversification events prevents aberrant chromosomal translocations and promote antibody diversification mechanisms. Recent advances in transcriptomics and genomics have identified the role of noncoding RNA RNA processing complex RNA exosome in the organization of B cell genome and regulation of the DNA mutator protein Activation Induced Deaminase (AID). In this application, we evaluate the role of RNA exosome complex and its cofactor proteins in regulating AID activity leading to robust class switch recombination and somatic hypermutation at the Immunoglobulin loci genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI099195-06
Application #
9310540
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Ferguson, Stacy E
Project Start
2012-05-07
Project End
2022-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
6
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Rothschild, Gerson; Basu, Uttiya (2017) Lingering Questions about Enhancer RNA and Enhancer Transcription-Coupled Genomic Instability. Trends Genet 33:143-154
Lim, Junghyun; Giri, Pankaj Kumar; Kazadi, David et al. (2017) Nuclear Proximity of Mtr4 to RNA Exosome Restricts DNA Mutational Asymmetry. Cell 169:523-537.e15
Rialdi, Alexander; Hultquist, Judd; Jimenez-Morales, David et al. (2017) The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity. Cell 169:679-692.e14
Laffleur, Brice; Basu, Uttiya; Lim, Junghyun (2017) RNA Exosome and Non-coding RNA-Coupled Mechanisms in AID-Mediated Genomic Alterations. J Mol Biol 429:3230-3241
Pefanis, Evangelos; Basu, Uttiya (2015) RNA Exosome Regulates AID DNA Mutator Activity in the B Cell Genome. Adv Immunol 127:257-308
Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson et al. (2015) RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 161:774-89
Sun, Jianbo; Wang, Jiguang; Pefanis, Evangelos et al. (2015) Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells. Cell Rep 13:1110-1117
Rothschild, Gerson; von Krusenstiern, A Nikolai; Basu, Uttiya (2015) Malaria-Induced B Cell Genomic Instability. Cell 162:697-8
Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson et al. (2014) Noncoding RNA transcription targets AID to divergently transcribed loci in B cells. Nature 514:389-93
Chao, Jaime; Rothschild, Gerson; Basu, Uttiya (2014) Ubiquitination events that regulate recombination of immunoglobulin Loci gene segments. Front Immunol 5:100

Showing the most recent 10 out of 13 publications