RAG Proteins Research Articles

The V(D)J recombination machinery is associated with the nuclear matrix (88.3)

Abstract Somatic recombination of immunoglobulin or T cell antigen receptor genes occurs through recombination activating gene product (RAG)-mediated cleavage at recombination signal sequences (RSS) and subsequently, the broken DNA ends are repaired by non-homologue end joining (NHEJ) enzymes. Here, we show that RAG1, RAG2, and many NHEJ factors in B lineage cells are associated with the nuclear matrix. The core RAG1 and RAG2 proteins have their own nuclear matrix targeting regions. RAG-mediated double stranded DNA breaks at the Jκ4 RSS can be readily detected in the nuclear matrix fraction in Gleevec treated Abelson transformed murine B cells or mouse bone marrow cells, indicating that the recombination reaction is ongoing on the nuclear matrix. In the HEK 293 cell-based recombination system, artificial recombination substrates are recruited to the nuclear matrix for recombination. Moreover, nuclear matrix proteins purified from 293 cells expressing the core RAG proteins or human B lineage cells expressing endogenous RAG proteins support cleavage of RSS substrates in vitro. Based on these results, we propose that the V(D)J recombination machinery is associated with the nuclear matrix.

Ragulator-Rag Complex Targets mTORC1 to the Lysosomal Surface and Is Necessary for Its Activation by Amino Acids

The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. We show that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which we term Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1.

Epigenetics Drives RAGs to Recombination Riches

V(D)J recombination of antigen receptor gene segments in B and T cells is mediated by the lymphoid-specific proteins RAG1 and RAG2. Now, Ji et al. (2010) demonstrate how RAG1 and RAG2 use DNA sequence specificity and modified histones within chromatin to target specific loci for V(D)J recombination at different stages of lymphoid development.

Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.

Postnatal Deamidation of 4E-BP2 in Brain Enhances Its Association with Raptor and Alters Kinetics of Excitatory Synaptic Transmission

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Requirement for ubiquitin conjugation and 26S proteasome activity at an early stage in V(D)J recombination

V(D)J recombination, the process that rearranges gene segments to assemble mature antigen receptor genes, relies on a recombinase comprising the RAG1 and RAG2 proteins. RAG1 is a multi-functional enzyme including DNA binding and cleavage as well as ubiquitin ligase activities, all of which appear to contribute to its role in recombination. Here we demonstrate that components of the ubiquitin conjugation machinery and the 26S proteasome are required for an early step in V(D)J recombination. Inhibitors of the 26S proteasome and ubiquitin activating enzyme (E1) blocked both chromosomal and extra-chromosomal recombination when added 1h following transfection/induction, but they had no effect when added 16h later. There was no effect on expression of RAG1, and recombination did not require transit through the cell cycle, confirming that inhibition was not due to an indirect effect on cell cycle arrest or protein expression. Experiments in which RAG1 translation was blocked with cyclohexamide after 16h of expression indicated that many active recombination complexes were formed within this window, although recombination products continued to accumulate for 48h. These data suggest that ubiquitin-dependent degradation is an early step in complex assembly or activation, and are consistent with our previous hypothesis that degradation of a negative regulator is required to trigger recombination.

Preliminary study on differential expression of plasma proteins of patients with Takayasu's arteritis in different stage

To investigate differential expression of plasma proteins of patients with Takayasu's arteritis to screen disease-related or phase-related proteins or biomarkers. From March, 2005 to January, 2006, Plasma of 20 patients with acute Takayasu's arteritis, 20 patients with chronic Takayasu's arteritis, and 20 healthy people as control were collected. Plasma proteins were profiled by two-dimensional electrophoresis. Spots of differential expression were screened by computerized map analysis and identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry. Circulation levels of parts of differential expression proteins were investigated by Elisa in each subject. Fourteen differential expression proteins were identified, including serum amyloid A, serum amyloid P, fibrinogen, complement C3c, C7, C4 binding protein, factor H related protein-1, immunoglobin, alpha-acid glycoprotein, RAG1 protein, alpha1-microglobin, apolipoprotein A-I, A-IV, transthyretin, haptoglobin. Proteins related to acute and chronic phase were serum amyloid A, fibrinogen, transthyretin, haptoglobin; Circulation levels of Serum amyloid A (SAA) and Complement C4 binding protein (C4BP) were significantly increased in active TA patients comparing to that in inactive TA patients and in controls (SAA: 95.9 vs 49.2, P = 0.009 and 23.9 mg/L, P = 0.001, respectively; C4BP: 88.5 vs 61.7, P = 0.023 and 32.6 mg/L, P < 0.001, respectively). Acute phase proteins and immune proteins may possible be markers for diagnosis and activity of Takayasu's arteritis, Complement activity, complement modulation protein and antibody production may be involved in immune mechanism of Takayasu's arteritis. Further study of these proteins may be helpful to elucidate the pathologic mechanism of Takayasu's arteritis.

The RING Domain of RAG1 Ubiquitylates Histone H3: A Novel Activity in Chromatin-Mediated Regulation of V(D)J Joining

The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination.

Organization of RAG1/2 and RSS DNA in the Post-Cleavage Complex

V(D)J recombination is central to establishing a functional adaptive immune system. The large repertoire of immunoglobulins and T-cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are joined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by collaboration of the lymphoid-specific proteins RAG1 and RAG2. After cleavage, the signal ends remain tightly bound to the RAG proteins in a particularly stable Signal-End Complex (SEC).To obtain 3D structural information about RAG1/2 bound to RSS DNA, isolated and purified SEC were visualized by AFM. To better define the arrangement of the RAG proteins and RSS DNA in the complex, we used RAG1 and RAG2 fused with maltose binding protein (MBP). A wide variety of complex shapes was recorded, however, it was clear that the two DNA chains predominantly exited the SEC complex from adjacent points. The volume of the protein core was consistent with the expected mass of 500 kDa corresponding to (RAG1)2-(RAG2)2 composition. MBP protrusions could be observed on the protein particles marking the N-termini of RAG1 and RAG2. To make their appearance more noticeable, we used selective antibody labeling. Fab-labeled MBPs were clearly identified peripheral to the recombinase core. When only the RAG2 MBPs were labeled, the two DNAs most often exited together from the SEC on the opposite side to the Fabs. Consistent with this observation, when only the RAG1 MBPs were labeled, they were situated closer to the exiting DNAs.The parallel arrangement of DNA and protein subunits found by AFM is in an excellent agreement with the 3D model based on EM data.

Unique features of NK cell development during ontogeny revealed in studies of RAG‐1‐deficient mice

atural killer (NK) cells link early innateimmune responses with adaptiveimmune responses. ‘Adaptive’ immune cells,that is, the T- and B-lymphocytes, expressclonally distributed receptors, the expressionof which is dependent on the recombinationactivation genes-1 (RAG-1) and RAG-2.Inthe absence of RAG-1 and/or RAG-2 proteins,T- and B-cell receptors cannot be formed andthe cells fail to undergo normal develop-ment.

Molecular analysis of T-B-NK+severe combined immunodeficiency and Omenn syndrome cases in Saudi Arabia

BackgroundChildren with Severe Combined Immunodeficiency (SCID) lack autologous T lymphocytes and present with multiple infections early in infancy. Omenn syndrome is characterized by the sole emergence of oligoclonal auto-reactive T lymphocytes, resulting in erythroderma and enteropathy. Omenn syndrome (OS) shares the genetic aetiology of T-B-NK+ SCID, with mutations in RAG1, RAG2, or DCLRE1C.MethodsPatients diagnosed with T-B-NK+ SCID or phenotypes suggestive of Omenn syndrome were investigated by molecular genetic studies using gene tightly linked microsatellite markers followed by direct sequencing of the coding regions and splice sites of the respective candidate genes.ResultsWe report the molecular genetic basis of T-B-NK+ SCID in 22 patients and of OS in seven patients all of Arab descent from Saudi Arabia. Among the SCID patients, six (from four families) displayed four homozygous missense mutations in RAG1 including V433M, R624H, R394W, and R559S. Another four patients (from three familes) showed 3 novel homozygous RAG2 mutations including K127X, S18X, and Q4X; all of which predict unique premature truncations of RAG2 protein. Among Omenn patients, four (from two families) have S401P and R396H mutations in RAG1, and a fifth patient has a novel I444M mutation in RAG2. Seven other patients (six SCID and one OS) showed a gross deletion in exons 1-3 in DCLRE1C. Altogether, mutations in RAG1/2 and DCLRE1C account for around 50% and 25%, respectively, in our study cohort, a proportion much higher than in previous reported series. Seven (24%) patients lack a known genetic aetiology, strongly suggesting that they carry mutations in novel genes associated with SCID and Omenn disorders that are yet to be discovered in the Saudi population.ConclusionMutation-free patients who lack a known genetic aetiology are likely to carry mutations in the regulatory elements in the SCID-causing genes or in novel genes that are yet to be discovered. Our efforts are underway to investigate this possibility by applying the whole genome scans on these cases via the use of Affymetrix high density DNA SNP chips in addition to homozygosity mapping.

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position -1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.

The Vam6 GEF Controls TORC1 by Activating the EGO Complex

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1(GTP), which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1(GDP), caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

RAG: a recombinase diversified

During B cell and T cell development, the lymphoid-specific proteins RAG-1 and RAG-2 act together to initiate the assembly of antigen receptor genes through a series of site-specific somatic DNA rearrangements that are collectively called variable-diversity-joining (V(D)J) recombination. In the past 20 years, a great deal has been learned about the enzymatic activities of the RAG-1-RAG-2 complex. Recent studies have identified several new and exciting regulatory functions of the RAG-1-RAG-2 complex. Here we discuss some of these functions and suggest that the RAG-1-RAG-2 complex nucleates a specialized subnuclear compartment that we call the 'V(D)J recombination factory'.

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Initial Stages of V(D)J Recombination: The Organization of RAG1/2 and RSS DNA in the Postcleavage Complex

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

BackgroundThe RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the “RAG transposon”.Methodology/Principal FindingsNovel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a “RAG transposon.” A subsequent “arms race” between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex).Conclusions/SignificanceA “co-regulatory” model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the “RAG-transposon” hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination.

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin‐6 to induce the expression of recombination‐activating gene in B lymphocytes

B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin kappa and lambda light chain expression and by ligation-mediated-PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the kappa-to-lambda ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA.

Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis

The products of recombination activating genes (RAG) 1 and 2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA-binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer-binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1/2.

The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

The enormous repertoire of the vertebrate specific immune system relies on the rearrangement of discrete gene segments into intact antigen receptor genes during the early stages of B-and T-cell development. This V(D)J recombination is initiated by a lymphoid-specific recombinase comprising the RAG1 and RAG2 proteins, which introduces double-strand breaks in the DNA adjacent to the coding segments. Much of the biochemical research into V(D)J recombination has focused on truncated or "core" fragments of RAG1 and RAG2, which lack approximately one third of the amino acids from each. However, genetic analyses of SCID and Omenn syndrome patients indicate that residues outside the cores are essential to normal immune development. This is in agreement with the striking degree of conservation across all vertebrate classes in certain non-core domains. Work from multiple laboratories has shed light on activities resident within these domains, including ubiquitin ligase activity and KPNA1 binding by the RING finger domain of RAG1 and the recognition of specific chromatin modifications as well as phosphoinositide binding by the PHD module of RAG2. In addition, elements outside of the cores are necessary for regulated protein expression and turnover. Here the current state of knowledge is reviewed regarding the non-core regions of RAG1 and RAG2 and how these findings contribute to our broader understanding of recombination.