Department of Microbiology & Immunology

Research Highlight

AID Targeted to Both DNA Strands by RNA Exosome: A Role for Non-coding RNA Surveillance Machinery in Antibody Diversity
Uttiya Basu, Assistant Professor of Microbiology & Immunology

Antigens that can elicit an immune response are almost infinite in number. However, there are only a very finite number of genes at our disposal to synthesize antibodies specific for each of these potentially harmful antigens. B lymphocytes resolve this seemingly insurmountable challenge by undergoing three impressive processes, each making a powerful contribution to the organism's ability to fend off attacks by potential pathogens.

First, immature B lymphocytes in the bone marrow undergo V(D)J recombination, which dramatically increases the immunoglobulin (Ig) repertoire of the organism. Then, these B cells migrate to secondary lymphoid organs, where they go through somatic hypermutation to increase the affinity of an immunoglobulin for the epitope it binds. Finally, they undergo class switch recombination (CSR) to diversify and best tailor the kind of effector function that results from interacting with an antigen. These last two genetic alterations, which establish antibody memory, absolutely require the activity of the ssDNA cytidine deaminase AID.

It is imperative to understand the mechanism of function of AID, since loss of AID activity leads to immune-deficiencies known as hyper IgM-syndrome, whereas hyper-AID activity initiates aberrant chromosomal lesions and translocations that lead to oncogenesis. In this regard, most B cell lymphomas are caused by chromosomal translocations of the Ig locus that lead to deregulated expression of protooncogenes. Furthermore, a large number of germinal center derived B-cell lymphomas are associated with hypermutation of proto-oncogenes at signature AID substrate motifs. Although the importance of AID as a key regulator of adaptive immunity and potential oncogene is well established, the molecular mechanism by which AID performs its function is not completely understood. How AID identifies its physiological target sequences in the B cell genome and imparts mutations on DNA is an active area of investigation.

In our recently published work we have implicated the cellular non-coding RNA-processing/degradation complex, RNA exosome, in targeting AID to both DNA strands. In B cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions and promotes optimal CSR. Moreover purified RNA exosome complex imparts robust AID- and transcription-dependent DNA deamination of both strands of transcribed DNA substrates in vitro, thus providing vital mechanistic insight into molecular mechanism of AID action. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity. Future work will focus on the role of non-coding RNAs and RNA exosome complex during generation of adaptive immune response using various mouse model systems.

Citation

Basu, U.*, Meng, F.L., Keim, C., Grinstein, V., Pefanis, E., Eccleston, J., Zhang, T., Myers, D., Wasserman, C.R., Wesemann, D.R., Januszyk, K., Gregory, R.I., Deng, H., Lima, C.D., Alt. F.W.*. (2011) The RNA Exosome Targets the AID Cytidine Deaminase to Both Strands of Transcribed Duplex DNA Substrates. Cell 144: 353-363. (*corresponding authors)

Recent Notable Faculty Publications

Minc, N., Burgess, D. and Chang, F. (2011) Influence of Cell Geometry on Division-Plane Positioning. Cell 144: 414-426. (A Cell Research Highlight)
Pasqualucci, L., Dominguez-Sola, D., Chiarenza, A., Fabbri, G., Grunn, A., Trifonov, V., Kasper, L.H., Lerach, S., Tang, H., Ma, J., Rossi, D., Chadburn, A., Murty, V.V., Mullighan, C.G., Gaidano, G., Rabadan, R., Brindle, P.K. and Dalla-Favera, R. (2011) Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature 471: 189-195.
Laaberki, M-H., Pfeffer, J., Clarke, A. and Dworkin J. (2011) O-acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis. J. Biol Chem 286: 5278-5288.
Adjalley, S.H., Johnston, G.L., Li, T., Eastman, R.T., Ekland, E.H., Eappen, A.G., Richman, A., Sim, B.K., Lee, M.C., Hoffman, S.L. and Fidock, D.A. (2011) Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue. Proc. Natl. Acad. Sci. U.S.A. Epub ahead of print 2011 Oct 31.
West, A.P., Brodsky, I.E., Rahner, C., Woo, D.K., Erdjument-Bromage, H., Tempst, P., Walsh, M.C., Choi, Y., Shadel, G.S. and Ghosh, S. (2011) TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472: 476-480.
Arriagada, G., Muntean, L.N. and Goff, S.P. (2011) SUMO-interacting motifs of human TRIM5alpha are important for antiviral activity. PLoS Pathog. 7:e1002019. Epub 2011 Apr 7.
Washburn, R.S. and Gottesman, M.E. (2011) Transcription termination maintains chromosome integrity. Proc. Natl. Acad. Sci. U.S.A. 108: 792-797.
Atarashi, K., Tanoue, T., Shima, T., Imaoka, A., Kuwahara, T., Momose, Y., Cheng, G., Yamasaki, S., Saito, T., Ohba, Y., Taniguchi, T., Takeda, K., Hori, S., Ivanov, I.I., Umesaki, Y., Itoh, K. and Honda, K. (2011) Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331: 337-341.
Klein, U. and Ghosh, S. (2011) The two faces of NF-kB signaling in cancer development and therapy. Cancer Cell. 20: 556-558.
Anandasabapathy, N., Victora, G.D., Meredith, M., Feder, R., Dong, B., Kluger, C., Yao, K., Dustin, M.L., Nussenzweig, M.C., Steinman, R.M. and Liu, K. (2011) Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain.J. Exp. Med. 208: 1695-1705. Epub 2011 Jul 25.
Rasmussen, A.L. and Racaniello, V.R. (2011) Selection of rhinovirus 1A variants adapted for growth in mouse lung epithelial cells. Virology 420: 82-88. Epub 2011 Sep 22.
Chang, J.T., Ciocca, M.L., Kinjyo, I., Palanivel, V.R., McClurkin, C.E., Dejong, C.S., Mooney, E.C., Kim, J.S., Steinel, N.C., Oliaro, J., Yin, C.C., Florea, B.I., Overkleeft, H.S., Berg, L.J., Russell, S.M., Koretzky, G.A., Jordan, M.S. and Reiner, S.L. (2011) Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division. Immunity 34: 492-504. Epub 2011 Apr 14.
Lewis, K.L., Caton, M.L., Bogunovic, M., Greter, M., Grajkowska, L.T., Ng, D., Klinakis, A., Charo, I.F., Jung, S., Gommerman, J.L., Ivanov, I.I., Liu, K., Merad, M. and Reizis, B. (2011) Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine. Immunity Epub ahead of print 2011 Oct 19.
Melillo, J.A., Song, L., Bhagat, G., Blazquez, A.B., Plumlee, C.R., Lee, C., Berin, C., Reizis, B. and Schindler, C. (2010) Dendritic cell (DC)-specific targeting reveals Stat3 as a negative regulator of DC function. J. Immunol. 184: 2638-2645.
Walters, M.S., Kinchington, P.R., Banfield, B.W. and Silverstein, S.J. (2010) Hyperphosphorylation of histone deacetylase 2 by alphaherpesvirus US3 kinases. J. Virol. 84: 9666-9676.
Lucas, C.L., Workman, C.J., Beyaz, S., LoCascio, S., Zhao, G., Vignali, D.A. and Sykes, M. (2011) LAG-3, TGF-beta, and cell-intrinsic PD-1 inhibitory pathways contribute to CD8 but not CD4 T-cell tolerance induced by allogeneic BMT with anti-CD40L. Blood. 117: 5532-5540. Epub 2011 Mar 21.
Ho, C.K., Mazon, G., Lam, A.F. and Symington, L.S. (2010) Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Molecular Cell 40: 988-1000.


Research Highlight AID Targeted to Both DNA Strands by RNA Exosome: A Role for Non-coding RNA Surveillance Machinery in Antibody Diversity Uttiya Basu, Assistant Professor of Microbiology & Immunology Antigens that can elicit an immune response are almost infinite in number. However, there are only a very finite number of genes at our disposal to synthesize antibodies specific for each of these potentially harmful antigens. B lymphocytes resolve this seemingly insurmountable challenge by undergoing three impressive processes, each making a powerful contribution to the organism's ability to fend off attacks by potential pathogens. First, immature B lymphocytes in the bone marrow undergo V(D)J recombination, which dramatically increases the immunoglobulin (Ig) repertoire of the organism. Then, these B cells migrate to secondary lymphoid organs, where they go through somatic hypermutation to increase the affinity of an immunoglobulin for the epitope it binds. Finally, they undergo class switch recombination (CSR) to diversify and best tailor the kind of effector function that results from interacting with an antigen. These last two genetic alterations, which establish antibody memory, absolutely require the activity of the ssDNA cytidine deaminase AID. It is imperative to understand the mechanism of function of AID, since loss of AID activity leads to immune-deficiencies known as hyper IgM-syndrome, whereas hyper-AID activity initiates aberrant chromosomal lesions and translocations that lead to oncogenesis. In this regard, most B cell lymphomas are caused by chromosomal translocations of the Ig locus that lead to deregulated expression of protooncogenes. Furthermore, a large number of germinal center derived B-cell lymphomas are associated with hypermutation of proto-oncogenes at signature AID substrate motifs. Although the importance of AID as a key regulator of adaptive immunity and potential oncogene is well established, the molecular mechanism by which AID performs its function is not completely understood. How AID identifies its physiological target sequences in the B cell genome and imparts mutations on DNA is an active area of investigation. In our recently published work we have implicated the cellular non-coding RNA-processing/degradation complex, RNA exosome, in targeting AID to both DNA strands. In B cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions and promotes optimal CSR. Moreover purified RNA exosome complex imparts robust AID- and transcription-dependent DNA deamination of both strands of transcribed DNA substrates in vitro, thus providing vital mechanistic insight into molecular mechanism of AID action. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity. Future work will focus on the role of non-coding RNAs and RNA exosome complex during generation of adaptive immune response using various mouse model systems. Citation *Basu, U.,** Meng, F.L., Keim, C., Grinstein, V., Pefanis, E., Eccleston, J., Zhang, T., Myers, D., Wasserman, C.R., Wesemann, D.R., Januszyk, K., Gregory, R.I., Deng, H., Lima, C.D., Alt. F.W.. (2011) The RNA Exosome Targets the AID Cytidine Deaminase to Both Strands of Transcribed Duplex DNA Substrates. Cell* 144: 353-363. (corresponding authors) Recent Notable Faculty Publications* Minc, N., Burgess, D. and Chang, F. (2011) Influence of Cell Geometry on Division-Plane Positioning. Cell 144: 414-426. (A Cell Research Highlight) Pasqualucci, L., Dominguez-Sola, D., Chiarenza, A., Fabbri, G., Grunn, A., Trifonov, V., Kasper, L.H., Lerach, S., Tang, H., Ma, J., Rossi, D., Chadburn, A., Murty, V.V., Mullighan, C.G., Gaidano, G., Rabadan, R., Brindle, P.K. and Dalla-Favera, R. (2011) Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature 471: 189-195. Laaberki, M-H., Pfeffer, J., Clarke, A. and Dworkin J. (2011) O-acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis. J. Biol Chem 286: 5278-5288. Adjalley, S.H., Johnston, G.L., Li, T., Eastman, R.T., Ekland, E.H., Eappen, A.G., Richman, A., Sim, B.K., Lee, M.C., Hoffman, S.L. and Fidock, D.A. (2011) Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue. Proc. Natl. Acad. Sci. U.S.A. Epub ahead of print 2011 Oct 31. West, A.P., Brodsky, I.E., Rahner, C., Woo, D.K., Erdjument-Bromage, H., Tempst, P., Walsh, M.C., Choi, Y., Shadel, G.S. and Ghosh, S. (2011) TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472: 476-480. Arriagada, G., Muntean, L.N. and Goff, S.P. (2011) SUMO-interacting motifs of human TRIM5alpha are important for antiviral activity. PLoS Pathog. 7:e1002019. Epub 2011 Apr 7. Washburn, R.S. and Gottesman, M.E. (2011) Transcription termination maintains chromosome integrity. Proc. Natl. Acad. Sci. U.S.A. 108: 792-797. Atarashi, K., Tanoue, T., Shima, T., Imaoka, A., Kuwahara, T., Momose, Y., Cheng, G., Yamasaki, S., Saito, T., Ohba, Y., Taniguchi, T., Takeda, K., Hori, S., Ivanov, I.I., Umesaki, Y., Itoh, K. and Honda, K. (2011) Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331: 337-341. Klein, U. and Ghosh, S. (2011) The two faces of NF-kB signaling in cancer development and therapy. Cancer Cell. 20: 556-558. Anandasabapathy, N., Victora, G.D., Meredith, M., Feder, R., Dong, B., Kluger, C., Yao, K., Dustin, M.L., Nussenzweig, M.C., Steinman, R.M. and Liu, K. (2011) Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain.J. Exp. Med. 208: 1695-1705. Epub 2011 Jul 25. Rasmussen, A.L. and Racaniello, V.R. (2011) Selection of rhinovirus 1A variants adapted for growth in mouse lung epithelial cells. Virology 420: 82-88. Epub 2011 Sep 22. Chang, J.T., Ciocca, M.L., Kinjyo, I., Palanivel, V.R., McClurkin, C.E., Dejong, C.S., Mooney, E.C., Kim, J.S., Steinel, N.C., Oliaro, J., Yin, C.C., Florea, B.I., Overkleeft, H.S., Berg, L.J., Russell, S.M., Koretzky, G.A., Jordan, M.S. and Reiner, S.L. (2011) Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division. Immunity 34: 492-504. Epub 2011 Apr 14. Lewis, K.L., Caton, M.L., Bogunovic, M., Greter, M., Grajkowska, L.T., Ng, D., Klinakis, A., Charo, I.F., Jung, S., Gommerman, J.L., Ivanov, I.I., Liu, K., Merad, M. and Reizis, B. (2011) Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine. Immunity Epub ahead of print 2011 Oct 19. Melillo, J.A., Song, L., Bhagat, G., Blazquez, A.B., Plumlee, C.R., Lee, C., Berin, C., Reizis, B. and Schindler, C. (2010) Dendritic cell (DC)-specific targeting reveals Stat3 as a negative regulator of DC function. J. Immunol. 184: 2638-2645. Walters, M.S., Kinchington, P.R., Banfield, B.W. and Silverstein, S.J. (2010) Hyperphosphorylation of histone deacetylase 2 by alphaherpesvirus US3 kinases. J. Virol. 84: 9666-9676. Lucas, C.L., Workman, C.J., Beyaz, S., LoCascio, S., Zhao, G., Vignali, D.A. and Sykes, M. (2011) LAG-3, TGF-beta, and cell-intrinsic PD-1 inhibitory pathways contribute to CD8 but not CD4 T-cell tolerance induced by allogeneic BMT with anti-CD40L. Blood. 117: 5532-5540. Epub 2011 Mar 21. Ho, C.K., Mazon, G., Lam, A.F. and Symington, L.S. (2010) Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Molecular Cell 40: 988-1000.		Graphical abstract highlighting targeting of AID to both strands of DNA by the RNA exosome cellular RNA processing/ degradation complex.