F Nina Papavasiliou

The Rockefeller University, New York City, New York, United States

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Publications (46)644.07 Total impact

  • Rebecca K Delker, F Nina Papavasiliou
    Nature Immunology 10/2013; 14(11):1112-4. · 26.20 Impact Factor
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    ABSTRACT: Beyond its well-characterized functions in antibody diversification, the cytidine deaminase AID can catalyze off-target DNA damage and has been hypothesized to edit RNA and mediate DNA demethylation. To comprehensively examine the effects of AID on the transcriptome and the pattern of DNA methylation ('methylome'), we analyzed AID-deficient (Aicda(-/-)), wild-type and AID-overexpressing activated B cells by high-throughput RNA sequencing (RNA-Seq) and reduced-representation bisulfite sequencing (RRBS). These analyses confirmed the known role of AID in immunoglobulin isotype switching and also demonstrated few other effects of AID on gene expression. Additionally, we detected no evidence of AID-dependent editing of mRNA or microRNA. Finally, the RRBS data did not support the proposed role for AID in regulating DNA methylation. Thus, despite evidence of its additional activities in other systems, antibody diversification seems to be the sole physiological function of AID in activated B cells.
    Nature Immunology 05/2013; · 26.20 Impact Factor
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    ABSTRACT: Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.
    Proceedings of the National Academy of Sciences 12/2012; · 9.81 Impact Factor
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    ABSTRACT: Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs), T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC). How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.
    PLoS Pathogens 08/2012; 8(8):e1002900. · 8.14 Impact Factor
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    ABSTRACT: Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.
    RNA 07/2011; 17(7):1296-306. · 5.09 Impact Factor
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    ABSTRACT: Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.
    Journal of Biological Chemistry 06/2011; 286(23):20366-74. · 4.65 Impact Factor
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    Laura Belver, F Nina Papavasiliou, Almudena R Ramiro
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    ABSTRACT: MicroRNAs (miRNAs) are a class of endogenous, non-coding regulatory RNAs that control gene regulation by guiding silencing protein complexes to mRNA in a sequence-dependent manner. In this way miRNAs are able to repress gene expression post-transcriptionally by affecting mRNA stability or translation. These ubiquitous molecules play central roles in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Within the context of the immune system, genetic studies have identified distinct roles for specific miRNAs in gene regulation during development, activation and maturation. Conversely, dysregulation of miRNA expression has been specifically correlated with cancer. This review outlines our current understanding of miRNA function in lymphocytes as it impacts expression of protein-coding genes in the context of proper development, as well as oncogenesis.
    Current opinion in immunology 02/2011; 23(3):368-73. · 10.88 Impact Factor
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    ABSTRACT: Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we used Rag1(-/-) (recombination activating gene 1(-/-)) blastocyst complementation and in vitro embryonic stem cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag1(-/-) chimeras displayed a dramatic reduction in thymic cellularity compared with control mice. Using embryonic stem cell differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag1(-/-) chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared with controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent, early-stage and distinct, Beclin 1-independent, late-stage processes.
    The Journal of Immunology 02/2011; 186(4):2201-9. · 5.52 Impact Factor
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    ABSTRACT: Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in the translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA sequencing (RNA-Seq) screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3' untranslated regions (3' UTRs). Further analysis established several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The transcriptomics approach to RNA editing presented here dramatically expands the list of APOBEC1 mRNA editing targets and reveals a novel cellular mechanism for the modification of transcript 3' UTRs.
    Nature Structural & Molecular Biology 02/2011; 18(2):230-6. · 11.90 Impact Factor
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    Brad R Rosenberg, Scott Dewell, F Nina Papavasiliou
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    ABSTRACT: RNA editing deaminases act on a variety of targets in different organisms. A number of such enzymes have been shown to act on mRNA, with the resultant nucleotide changes modifying a transcript's information content. Though the deaminase activity of mRNA editing enzymes is readily demonstrated in vitro, identifying their physiological targets has proved challenging. Recent advances in ultra high-throughput sequencing technologies have allowed for whole transcriptome sequencing and expression profiling (RNA-Seq). We have developed a system to identify novel mRNA editing deamination targets based on comparative analysis of RNA-Seq data. The efficacy and utility of this approach is demonstrated for APOBEC1, a cytidine deaminase with a known and well-characterized mRNA editing target in the mammalian small intestine.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 718:103-19. · 1.29 Impact Factor
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    Pete Stavropoulos, F Nina Papavasiliou
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    ABSTRACT: The African trypanosome (Trypanosoma brucei) is transmitted by the bite of the tsetse vector to the mammalian bloodstream where it exists as a completely extracellular parasite. As a result of this exposure, the parasite elicits a robust immune response that is almost exclusively antibody mediated, and is extremely specific to the trypanosome coat displayed on the surface. This coat is comprised of ~11 million copies of a single gpi-linked molecule (the variable surface glycoprotein or VSG) and can therefore be used as a powerful platform for the immunogenic display of antigenic determinants. Here we describe a method to display repetitive, ordered arrays of linear epitopes on the surface of T. brucei and to then use the engineered organisms to generate specific anti-epitope antibody responses, upon injection into mice. This method offers an alternative approach to generating anti-peptide antibodies, and could be a useful option in cases where more traditional methods have failed.
    Journal of immunological methods 10/2010; 362(1-2):190-4. · 2.35 Impact Factor
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    Eric L Fritz, F Nina Papavasiliou
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    ABSTRACT: The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field.
    Genes & development 10/2010; 24(19):2107-14. · 12.08 Impact Factor
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    Claire E Hamilton, F Nina Papavasiliou, Brad R Rosenberg
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    ABSTRACT: Polynucleotide DNA and RNA editing enzymes alter nucleic acid sequences and can thereby modify encoded informational content. Two major families of polynucleotide editing enzymes, the AID/APOBEC cytidine deaminases (which catalyze the deamination of cytidine to uridine) and the adenosine deaminases acting on RNA (ADARs, which catalyze the deamination of adenosine to inosine), function in a variety of host defense mechanisms. These enzymes act in innate and adaptive immune pathways, with both host and pathogen targets. DNA editing by the cytidine deaminase AID mediates immunoglobulin somatic hypermutation and class switch recombination, providing the antibody response with the flexibility and diversity to defend against an almost limitless array of varied and rapidly adapting pathogenic challenges. Other cytidine deaminases (APOBEC3) restrict retroviral infection by editing viral retrogenomes. Adenosine deaminases (ADARs) shape innate immune responses by modifying host transcripts that encode immune effectors and their regulators. Here we review current knowledge of polynucleotide DNA and RNA editors with a focus on these and other functions they serve in the immune system.
    RNA biology 03/2010; 7(2):220-8. · 5.56 Impact Factor
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    Jan Davidson-Moncada, F Nina Papavasiliou, Wayne Tam
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    ABSTRACT: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to complementary target mRNAs and either promoting their decay or inhibiting their translation. Most eukaryotic genomes studied encode miRNAs, which are processed from longer noncoding transcripts through pathways conserved from fungi to plants to animals. miRNAs are now understood to be key mediators of developmental transitions in a number of model organisms. With respect to the immune system, miRNAs affect all facets of immune system development, from hematopoiesis to activation in response to infection during both the innate and the adaptive immune response. At the same time, miRNA dysregulation is a central event in the development and pathophysiology of a number of cancers of the immune system. Here we will discuss our current understanding of this general regulatory mechanism, focusing on its involvement in inflammation and in oncogenesis.
    Annals of the New York Academy of Sciences 01/2010; 1183:183-94. · 4.38 Impact Factor
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    ABSTRACT: Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3' enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3' heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3' enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.
    Journal of Experimental Medicine 11/2009; 206(12):2613-23. · 13.21 Impact Factor
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    Rebecca K Delker, Sebastian D Fugmann, F Nina Papavasiliou
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    ABSTRACT: The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implications of AID function on human physiology. We also take time to point out important ongoing controversies and outstanding questions in the field and highlight key experiments and techniques that hold the potential to elucidate the remaining mysteries surrounding this vital protein.
    Nature Immunology 11/2009; 10(11):1147-53. · 26.20 Impact Factor
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    ABSTRACT: Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.
    Nature 05/2009; 459(7244):278-81. · 38.60 Impact Factor
  • Grace Teng, F Nina Papavasiliou
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    ABSTRACT: Noncoding RNAs (ncRNAs), both small and large, have recently risen to prominence as surprisingly versatile regulators of gene expression. In fact, eukaryotic transcriptomes are rife with RNAs that do not code for protein, though the majority of these species remains wholly uncharacterized. The functional diversity among the mere handful of validated ncRNAs hints at the vast regulatory potential of these silent biomolecules. Though the act of noncoding transcription and the resultant ncRNAs do not directly produce proteins, they represent powerful means of gene control. Here we survey the accumulating literature on the myriad functions of long ncRNAs and emphasize one curious case of noncoding transcription at antigen receptor loci in lymphocytes.
    Advances in Immunology 01/2009; 104:25-50. · 7.26 Impact Factor
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    Grace Teng, F Nina Papavasiliou
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    ABSTRACT: Small RNAs mediate a diverse pot-pourri of post-transcriptional silencing mechanisms, ranging from 'classical' RNA interference (RNAi), to gene repression by microRNAs (miRNAs), to maintenance of genomic stability by repeat-associated small RNAs. Here, we review recent findings on the function of miR-155, particularly its roles in mammalian innate and adaptive immunity, viral infection and oncogenesis.
    Philosophical Transactions of The Royal Society B Biological Sciences 12/2008; 364(1517):631-7. · 6.23 Impact Factor
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    ABSTRACT: B lymphocytes perform somatic hypermutation and class-switch recombination (CSR) of the immunoglobulin locus to generate an antibody repertoire diverse in both affinity and function. These somatic diversification processes are catalyzed by activation-induced cytidine deaminase (AID), a potent DNA mutator whose expression and function are highly regulated. Here we show that AID was regulated posttranscriptionally by a lymphocyte-specific microRNA, miR-155. We found that miR-155 was upregulated in murine B lymphocytes undergoing CSR and that it targeted a conserved site in the 3'-untranslated region of the mRNA encoding AID. Disruption of this target site in vivo resulted in quantitative and temporal deregulation of AID expression, along with functional consequences for CSR and affinity maturation. Thus, miR-155, which has recently been shown to play important roles in regulating the germinal-center reaction, does so in part by directly downmodulating AID expression.
    Immunity 06/2008; 28(5):621-9. · 19.80 Impact Factor

Publication Stats

4k Citations
644.07 Total Impact Points

Institutions

  • 1995–2013
    • The Rockefeller University
      • • Laboratory of Lymphocyte Biology
      • • Laboratory of RNA Molecular Biology
      • • Laboratory of Molecular Immunology
      New York City, New York, United States
  • 2007–2011
    • The Ohio State University
      • Department of Microbiology
      Columbus, OH, United States
    • University of Rochester
      • Department of Biochemistry and Biophysics
      Rochester, NY, United States
  • 2009
    • University of Michigan
      • Department of Microbiology and Immunology
      Ann Arbor, MI, United States
    • Yale University
      • Department of Immunobiology
      New Haven, CT, United States
  • 2000
    • Howard Hughes Medical Institute
      Maryland, United States