Occurring N-Terminal Truncated RAG1 IsoformsA Spontaneous RAG1 Nonsense Mutation Unveils Naturally

Bassing and Edward M. BehrensThomas N. Burn, Kyutae D. Lee, Noor Dawany, Tanner F. Robertson, Megan R. Fisher, Craig H.

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2020, 4 (3) 119-128ImmunoHorizons 

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A Spontaneous RAG1 Nonsense Mutation Unveils NaturallyOccurring N-Terminal Truncated RAG1 Isoforms

Thomas N. Burn,*,† Kyutae D. Lee,‡ Noor Dawany,§ Tanner F. Robertson,*,‡ Megan R. Fisher,*,‡ Craig H. Bassing,*,‡ andEdward M. Behrens*,†

*Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; †Division of Rheumatology, The

Children’s Hospital of Philadelphia, Philadelphia, PA 19104; ‡Department of Pathology and Laboratory Medicine, The Children’s Hospital of

Philadelphia, Philadelphia, PA 19104; and §Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia,

PA 19104

ABSTRACT

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination,

allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus

of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous

nonsense mutation in the 59 region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal

translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects

in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 59 RAG1 frameshift mutations. We show

that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1

mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1

and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of

59 nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting

nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

ImmunoHorizons, 2020, 4: 119–128.

INTRODUCTION

The RAG1/RAG2 hetero-tetramer has been extensively studied inits role as the enzyme responsible for rearranging Ag receptorgenes in B andT cells in the process known as VDJ recombination(1, 2). RAG1 contains the catalytic domain necessary for DNAcleavage for this process, and thus, patients with loss-of-functionmutations in either RAG1 or RAG2 suffer from SCIDwith completeloss of mature T and B cells. In contrast, RAG1/2 mutations that

reduce but do not ablate recombination activity lead to Omennsyndrome (OS), an immunodeficiency of low B and T cell numbers,concurrentwith autoimmunity (3, 4).Notably, Santagata et al. (5)described patientswith homozygous frameshiftmutations in theN-terminal region of RAG1 that result in early translation termi-nation and should not yield functional RAG1 protein. Despite this,these patients had low but detectable circulating B and T cells.RAG1 is translated from a single exon; thus alternative splicingwas not considered. It was hypothesized that N-truncated RAG1

Received for publication January 3, 2020. Accepted for publication February 11, 2020.

Address correspondence and reprint requests to: Dr. Edward M. Behrens, Children’s Hospital of Philadelphia, Abramson Research Center Room 1102, 3615 CivicCenter Boulevard, Philadelphia, PA 19104-4399. E-mail address: behrens@email.chop.edu

ORCIDs: 0000-0003-1763-1418 (T.N.B.); 0000-0003-2983-3893 (N.D.); 0000-0003-1703-0786 (C.H.B.).

This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute Grant R01 HL112836-A1 (to E.M.B.); NIH/National Institute ofAllergy and Infectious Diseases Grant R01 AI121250-A1 (to E.M.B.); and the Nancy Taylor Foundation for Chronic Disease (to E.M.B.).

Abbreviations used in this article: ABC, age-related B cell; ANA, anti-nuclear Ab; DP, double-positive; iM, initiating methionine; OS, Omenn syndrome; qPCR,quantitative PCR; SP, single-positive; TIS, translation initiation site; Treg, T regulatory cell; WT, wild-type.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license.

Copyright © 2020 The Authors

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RESEARCH ARTICLE

Adaptive Immunity

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products arise from internal translation initiation at downstreamAUG start sites, leading to a hypomorphic protein. Correlativewith this hypothesis, expression of N-truncated cDNA gives riseto RAG1 proteins of smaller size that catalyze VDJ reactions invitro. However, neither the mechanism bywhich these truncatedRAG1 proteins are made nor whether they occur normally wereelucidated (5).

During a routine analysis, we identified a strain of mice in ourcolony with reduced T and B cells and increased activated T cellfrequencies. Whole-exome sequencing revealed a novel homozy-gous nonsense mutation at aa 60 (Q60X), which we termed theN-terminal stop or RAG1NX mouse. This mutation, like frameshiftmutations present in OS patients, would be predicted to causepremature translation termination and absence of functionalRAG1. However, the presence of T and B cells in these mice,although at lower numbers, necessitated the presence of resid-ual RAG1 activity. In this study, we show that the Q60X mutationin RAG1NX thymocytes results in expression of smaller RAG1isoforms. Similarly sized smaller RAG1 isoforms are present inhomozygous RAG1WT thymocytes. Further, in vitro experimentssuggest that these smaller RAG1 isoforms are generated usinginternal translation initiation sites (TIS). Our data demonstratethat an underappreciatedmechanism of internal translation likelyleads to multiple RAG1 isoforms in wild-type (WT) mice andallows for the escape from early truncating mutations in the genethat would otherwise have devastating immune consequences.There is significant evidence that the N-terminal regions of RAG1have important roles in VDJ recombination (6–10), and RAG1has been studied as a single isoform, containing the completeN-terminal region for over 30 y. The conceptual advance that mul-tipleN-truncatedRAG1isoformsexist, even inWTcells, is importantas theseN-truncatedproteinsdemonstratealteredfunctionandhavenot previously been interrogated as part of RAG biology. This novelmurine model of OS provides an important platform to deter-mine howN-truncated RAG1 proteins function normally and in theabsence of full-length RAG1 protein.

MATERIALS AND METHODS

MiceC57BL/6-RAG1WT mice were purchased from The Jackson Lab-oratory and bred in our facility. C57BL/6-RAG1NX and -RAG1Het

mice were bred in our facility. All animal studies were performedwith the approval of The Children’s Hospital of PhiladelphiaInstitutional Animal Care and Use Committee.

Whole-exome analysis and Sanger sequencingGenomic DNA was isolated from an RAG1NX spleen using theDNeasyBlood andTissueKit (Qiagen) permanufacturer’s instruc-tions. Exon capture for whole-exome sequencing was performedusing the Agilent SureSelect XT Mouse All Exon kit. Sequencingwas performed on the Illumina HiSeq 4000 to produce 150-bppaired-end reads with an average depth 3100. Sequence readswere aligned to the reference mouse genome (GRCm38/mm10)

using Novoalign (V3.03.01; http://www.novocraft.com). Picardwas used for marking duplicates, and then variants were calledusing GATK’s HaplotypeCaller. Single-nucleotide variants andinsertions/deletions were functionally annotated with SnpEff(http://snpeff.sourceforge.net) andfiltered to retainonlymoderate-and high-effect variants. After applying quality filters and exclud-ing variants that have been reported in the Single NucleotidePolymorphism Database, 299 variants remained. Genes werethen annotated using gene ontologies to identify variants ingenes involved in B and T cell development. A homozygous stopgain mutation was identified in Rag1 (NM_009019.2; p.Gln60*)that met all filtering and quality-control criteria.

Flow cytometric analysisSplenocytes, thymocytes, andbonemarrowleukocyteswerestainedwith LIVE/DEAD fixable viability dye (Life Technologies) and Absagainst respective surface Ags (BD Pharmingen, eBioscience, andBioLegend). For intracellular HA, FLAG, GFP, and Foxp3 staining,cells were stained using the eBioscience Foxp3 kit accordingto manufacturer’s instructions. All samples were acquired on anMACSQuantflowcytometer (MiltenyiBiotec) orLSRIIFortessa (BDBiosciences) andanalyzedusingFlowJosoftwareversion 10.5.3 (TreeStar).

Intracellular cytokine stainingSplenocytes (106)werecultured in theabsenceorpresenceof50ng/ml PMA (Sigma-Aldrich) and 1 mg/ml ionomycin (Cell SignalingTechnology), with 2 mg/ml brefeldin A (Sigma-Aldrich) and 2 mMmonensin (eBioscience) for 5 h at 37°C. After staining for LIVE/DEADandfor surfaceAgsasdescribedearlier, cellswere stained forthe respective cytokines using the Cytofix/Cytoperm kit accordingto manufacturer’s instructions (BD Bioscience).

Quantitative PCR of RAG1Relevant thymocyte populations were stained as described above,and sorted using a FACSAria Fusion. RNA was isolated usingRNeasy Mini kit (Qiagen) and reverse transcribed to cDNA usingSuperScript III Reverse Transcriptase (Invitrogen) with randomhexamers. Quantitative PCR (qPCR) was performed using PowerSYBR Green (Applied Biosystems), and HPRT or RAG1 primers(Qiagen).

qPCR of VDJ rearrangements in DN3 thymocytesThymocyteswere isolated, thenstainedwithPE-labeledanti-CD4,CD8, CD11b, CD11c, NK1.1, Gr1, and Ter119. Nonlabeled cells wereenriched by MACS depletion using anti-PE microbeads and LScolumns (Miltenyi). Enriched cells were then stained with CD4,CD8, CD44, and CD25. DN3 cells were sorted using a FACSAriaFusion. Genomic DNA was extracted from the sorted DN3 cellsusing the DNeasy Blood and Tissue Kit (QIAGEN). A qPCR assayto measure Vb-Db1-Jb1 and Vb-Db2-Jb2 rearrangement fre-quencies was designed with a panel of primers specific for eachfunctional Vb paired with a probe (FAM, HEX) specific for eitherJb1.1 or Jb2.1, respectively. Rearrangements were measured byTaqMan qPCRwith PCR conditions according to the manufacturer’s

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instructions (PrimeTime; Integrated DNA Technologies). PCRanalysis of TRDV1-JD1 and CD19 were used for normalization.Primers and probes have been described previously (11).

Anti-nuclear AbsHep2 anti-nuclear Ab (ANA) slides (MBL International) wereincubatedwithmouse serum diluted 100-fold in PBS for 15 min inthe dark. Slides were washed with PBS and stained with DAPI(Thermo Fisher Scientific) and anti-mouse Ig-AF488 (JacksonImmunoResearch Laboratories). Images were acquired on a203 plan apochromat and 1.4 numerical aperture objective on aspinning disc confocal system (UltraView ERS 6; PerkinElmer,Waltham, MA) equipped with an ORCA-ER camera (Hama-matsu Photonics, Bridgewater, NJ) and velocity software (v6.1.1;PerkinElmer). Instrument settings were fixed for all images, andresearchers were blinded to the sample identification. NuclearAF488 intensitywas quantified using FIJI software, usingDAPI todesignate the nuclear region of interest. A minimum of 30 nucleiwere analyzed per well, and the average AF488 intensity wasreported.

Cloning and RAG1 mutagenesisFLAG-RAG1-HA was amplified by PCR using the murineRAG1-HA template from the Bassing laboratory and cloned intothe NotI and XhoI sites of pZHK:CMV-IRES-GFP (12) to create thebase pZHK:CMV-FLAG-RAG1(WT)-HA-IRES-GFP construct. PCRwere performed using Takara ex-taq DNA polymerase (Takara).

The RAG1 cloning primers were as follows: forward: 59-TGAGCGGGTACCCAATTGGCCAATTGGGATCCGCGG-39 andreverse: 59-TGAGCGGTCGACCAATTGGCCAATTGCTCGAGTCTA-39.

For mutagenesis of methionines and Q60X, RAG1 wassubcloned into pUC18, and mutagenesis was performed usingQuikchange Lightning Kit (Agilent). Mutagenesis primers weredesigned using the online Quikchange Primer Design software(Agilent). Correctly mutated fragments were swapped back intothe pZHK vector using NotI and XhoI sites for expression.

Transfection of 293T cells293T cells were grown in six-well plates to 70–80% confluency.293T cells were transfected with 2 mg of the respective RAG1constructs using TransIT-Lenti reagent (Mirus Bio) according tomanufacturer’s instructions. Cells were washed 23 with PBS16–20 h posttransfection for flow cytometry or Western blot.

SDS-PAGE and Western blottingNative RAG1 Western blotting was performed as previously de-scribed (13) using rabbit anti-mouse RAG1 Ab from David Schatz(Yale). For RAG1 expressed in 293T cells, transfected cells werelysed usingM-PER lysis buffer (Thermo Fisher Scientific) withprotease inhibitor mixture (Thermo Fisher Scientific). Fifteenmicrograms of protein was boiled in loading buffer with 5%b-MEfor 10minand runona4–12%SDS-PAGEgel before transferto nitrocellulose membrane (Bio-Rad). Membranes were probedwith anti-HA (Clone 3F10; Sigma-Aldrich) and anti-FLAG (Clone

M2; Sigma-Aldrich) and then AF700 and AF780 conjugated sec-ondary Abs (Invitrogen) before imaging on a Licor Odyssey.

Statistical analysisAll data were analyzed in GraphPad Prism 8 using statisticaltests indicated in the figure legends. Error bars indicatemean6 SEM. The p values, 0.05 are considered significant(*p , 0.05, **p , 0.01, ***p , 0.0001, and ****p , 0.0001).A p value . 0.05 was not significant.

RESULTS

Novel nonsense mutation identified in 59 region of Rag1 geneRoutine analysis of PBMCs in a line of C57BL/6 mice within ourcolony revealed B and T cell lymphopenia of unknown cause(data not shown). Whole-exome sequencing of these mice wasperformed, and 1386 de novomutations were identified comparedwith the reference C56BL/6 genome (mm10). A homozygousnonsense mutation early in the Rag1 gene at nucleotide position187 (c187C . T) was identified as a mutation likely to drive theobserved phenotype. This mutation was confirmed by Sangersequencing (Fig. 1A, 1B). Thesemicewere backcrossedaminimumof three generations to WT C57BL/6 mice acquired from TheJackson Laboratory while selecting for the mutant RAG1 allele todecrease the likelihood that the other de novo mutations couldinfluence theobservedphenotype.Thismutation introduces a stopcodon that is predicted to terminate translation at aa 60 of RAG1(Q60X) and result in no functional RAG1 (Fig. 1A, 1B). We namedthis the RAG1NX mouse. However, unlike complete RAG1 defi-ciency, this homozygous mutation does not result in completeabsence of T and B cells. We therefore checked for RAG1 mRNAby qPCR and RAG1 protein by Western blot from homozygousRAG1NX thymocytes and show an increase in RAG1mRNA and theformation of a number of proteins of smaller sizes than full-length,WT RAG1 (Fig. 1C, 1D). Truncating frameshift mutations in the59 region of the Rag1 gene in human patients are unexpectedlyassociated with OS rather than SCID (5). Thus, we considered thatthe RAG1NX mouse may represent a model OS to investigate howsuch mutations retain enough RAG activity to prevent completeSCID.

RAG1NX mice have lymphocyte developmental blocks at Agreceptor rearrangement stepsGiven the mutation in RAG1, we sought to determine the effecton T and B cell development. RAG1NX thymuses demonstrate asignificant reduction in total cellularity, accounted for by a;3-foldreduction in the number of double-positive (DP) (CD4+CD8+)and mature single-positive (SP) cells (Fig. 2A, 2B). The majordevelopmental block occurs at the DN3 to DN4 transition (Fig.2C, 2D). We find an analogous block in B cell development, atthe pro-B to pre-B transition (Fig. 2E, 2F). These phenotypesare inherited in an autosomal recessive manner, as RAG1NX

heterozygotesarephenotypically indistinguishable fromhomozygousRag1WT mice.

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The DN3-DN4 transition for developing T cells and pro-B topre-B transition for B cells require the productive rearrangementof the TCRb allele or IgH chain for T and B cells, respectively, andsubsequent signaling through either the pre-TCRor pre-BCR (14).Blocks at these developmental stages can be due to defective re-combination efficiency; therefore, we sought to determinewhetherRAG1NX cells had decreased VDJ recombination.

RAG1NX thymocytes have reduced TCRVb recombinationand an altered Vb TCR repertoireTo examine recombination efficiency and Vb repertoire inRAG1NXmice,we sortedDN3 thymocytes and assayedVb-DbJbrearrangements by TaqMan PCR. We find substantially lower-than-normal levels of rearrangements of all Vb gene segmentsin RAG1NX DN3 cells (Fig. 3A, 3B). We also determined the Vb

FIGURE 1. Novel nonsense mutation identified in 59 region of Rag1 gene.

(A) Diagram of RAG1 protein showing relative position of novel nonsense mutation (Q60X). (B) Sanger sequencing tracks aligning sequences from

RAG1WT and RAG1NX mice in our colony to that of the reference mm10 genome (National Center for Biotechnology Information). (C) DN3

thymocytes (live singlets, CD42, CD82, CD25+, CD442) and preselected DP thymocytes (live singlets, CD4+, CD8+, CD692) were sorted from

RAG1WT and RAG1NX mice. RNA was isolated and converted to cDNA. Relative RAG1 expression was measured by the DDCt method using the

housekeeping gene HPRT and a WT sample as a calibrator. Analyzed by two-way ANOVA with Tukey honest significant difference posttest. (D) RAG1

protein expressed in bulk thymocytes was analyzed by SDS-PAGE and Western blot. **p , 0.01, ***p , 0.0001.

FIGURE 2. RAG1NX mice have lymphocyte developmental blocks at Ag receptor rearrangement steps.

Thymi from 4- to 5-wk-old WT, NX, and Het littermates were analyzed by flow cytometry. (A) CD4 versus CD8 frequencies and (B) total cell counts

per thymus. Gating: live, singlets, dump2 (B220, CD11b, CD11c, Gr1, NK1.1, Ter119), TCRgd2. (C) DN1-4 frequencies and (D) total cell counts per

thymus. Gating: CD42, CD82, TCRblo. Early B cell development was analyzed in the bone marrow. (E and F) Frequencies of early B cell progenitors

of B220+CD93+IgM2 cells were quantified. Pregating: live, singlets, dump2 (TCRb, NK1.1, Ter119, CD11c, Gr1). Data combined from at least three

independent experiments. Bars indicate mean 6 SEM. Statistics: one-way ANOVA with Tukey honest significant difference posttest. **p , 0.01,

****p , 0.0001. n.s., p . 0.05.

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repertoireof thymocytes thatproducea functionalTCRbbysurfacestaining and show that there is an altered repertoire at both DPand SP stages (Fig. 3C, 3D, Supplemental Fig. 1A, 1B). Finally, byanalyzing the ratio of Vb usage at the DP versus the SP stage, weshow that there is altered selection of certain Vb proteins, suchas less selection of Vb4 and greater selection of Vb5 (Fig. 2E).Correspondingly, Vb usage by bulk thymocytes is altered asevidenced by TaqMan PCR of Vb-DbJb rearrangements on totalthymocytes (Supplemental Fig. 1C, 1D).

RAG1NX mice have reduced numbers of mature B and T cellsand increased frequencies of T regulatory andT memory cellsRAG1NX mice have an;50% reduction in peripheral T and B cellcounts. This phenotype is inherited in an autosomal recessivemanner, as RAG1NX heterozygotes are phenotypically indistin-guishable from homozygous Rag1WT mice (Fig. 4A). There is noeffect on dendritic cells, neutrophils, and inflammatory mono-cytes, but there is a slight increase in the numbers of splenic NKcells (Fig. 4A). RAG1NXmice had higher frequencies of T regulatorycells (Tregs) (Fig. 4B) and memory phenotype CD4+ and CD8+

T cells (Fig. 4C, 4D). The total numbers of Tregs and memoryphenotype cells were equivalent across genotypes (Fig. 4B,4D). This phenotype is consistent with the leaky-SCID/OSphenotype observed in humans and other mouse models ofOS (3, 15–17).

RAG1NX mice develop increased age-associated B cellsand ANAGiven the similarity of the Q60X mutation present in RAG1NX

mice to lesions found in a number of OS patients, we assessedphenotypes typically associated with development of autoimmu-nity (5). Ex vivo–stimulated CD4+ T cells from young (4- to 6-wk-old) RAG1NXmice showaheightenedpropensity to express IFN-gand IL-17A, andCD8+T cells expressmore IFN-g andTNF-a thanWT counterparts (Fig. 5A, 5D). OS patients often present witherythroderma, eosinophilia, high circulating IgE levels, and Th2skewing, including T cell IL-4 and IL-5 production. Although it isclear that young RAG1NX mice preferentially adopt a Th1-skewedT cell phenotype, more characteristic of the C57BL/6 strain (18),we were interested to see whether canonical OS phenotypesarose as the mice aged. RAG1NX mice aged 20–22 mo have nodifference in immune cell populations, and T and B cell countshave equilibrated inmiceby this age (Fig. 5E).There is no evidenceof increased eosinophils. Overall RAG1NX mice have no overtautoimmune pathology (data not shown) but feature increasedCD11b+CD11c+ age-relatedB cells (ABCs) (Fig. 5F). ABCs are oftenassociated with a predisposition toward ANA-positive autoim-mune diseases (19). Accordingly, aged RAG1NX mice have higherANA levels (Fig. 5G, Supplemental Fig. 2). Overall, these datasuggest that although RAG1NX mice do not have overt OS diseaseas seen in humans, they may have a predisposition towardautoimmunity.

FIGURE 3. RAG1NX thymocytes have reduced TCRVb recombination and an altered Vb TCR repertoire.

Sorted DN3 thymocytes from WT and NX mice were assayed by qPCR for frequencies of respective Vb to DbJb1 rearrangements (A) and Vb to

DbJb2 rearrangements (B), four mice per genotype. A number sign (#) denotes not detected. Gating: live, singlets, dump2 (B220, CD8, CD4, CD11b,

CD11c, Gr1, NK1.1, Ter119), CD42, CD82, CD25+, CD442. Vb repertoire was assayed by flow cytometry of (C) DP thymocytes and (D) SP thymocytes.

Representative gating in Supplemental Fig. 1A, 1B. (E) The ratio of Vb usage by DP versus SP thymocytes was calculated. Data combined from two

independent experiments. Error bars indicate SEM. Statistics: multiple t tests with Holm-Sidak correction. *p , 0.05, **p , 0.01, ***p , 0.0001,

****p , 0.0001. n.s., p . 0.05.

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Smaller RAG1 isoforms arise via internaltranslation initiationGiven the position of the RAG1NX nonsensemutation (Fig. 1A) andthe presence of smaller-than-normal RAG1 proteins in RAG1NX

cells (Fig. 1C), we were intrigued as to how these smaller proteinsform. The description of OS patients with 59 RAG1 frameshiftmutations hypothesized that alternate N-truncated isoforms arisevia translation from alternative TIS (5). Although this reportshowed that N-terminally truncated RAG1 proteins function inrecombination assays, whether alternative translation initiationcreates these isoforms was not directly assessed. Interestingly,even RAG1WT thymocytes have expression of smaller proteins(Fig. 1C). These proteins have been noted and designated previ-ously as “break-down products” (20). We hypothesized that these

smaller proteins are naturally occurring isoforms created fromtranslation of alternative TIS. To test this, we created RAG1 pro-teins epitope tagged at N-terminal (FLAG) and C-terminal (HA)ends. Probing for FLAG only detects full-length RAG1 translatedfrom the canonical TIS, whereas probing for HA would reveal allpossible internally translated isoforms (Fig. 6A). Site-directedmutagenesis was performed to introduce the Q60X mutation andtomutate the canonical initiatingmethionine (iM) codon, AUG, toisoleucine (AUC, iMI). RAG1-WT, RAG1-Q60X, and RAG1-iMIconstructs were transfected into 293T cells, and FLAG and HAexpressionwas analyzed. FLAG expression is only detected in theWT-transfected cells (Fig. 6B), whereas the C-terminal HA tag isdetected in all RAG1-transfected cells (Fig. 6C). The full-lengthband at;120 kDa is detected by the FLAG tag inWT byWestern

FIGURE 4. RAG1NX mice have reduced numbers of mature B and T cells and increased frequencies of Tregs and T memory cells.

(A) Numbers of leukocytes were enumerated from spleens of 4- to 5-wk-old RAG1WT (WT), RAG1NX (NX), or heterozygous (Het) littermates by flow

cytometry. Relative frequencies and numbers of peripheral Tregs (B), memory CD4+ T cells (C), and memory CD8+ T cells (D) in the spleen were

quantified. Data combined from at least three independent experiments. Bars indicate mean 6 SEM. Statistics: one-way ANOVA with Tukey honest

significant difference posttest. Gating: live, singlets, B cells (CD19+TCRb2), T cells (TCRb+, CD192, CD4+, or CD8+), NK cells (TCRb2, CD192,

NK1.1+), dendritic cells (TCRb2, CD192, NK1.12, CD11c+), neutrophils (TCRb2, CD192, NK1.12, CD11c2, CD11b+, Ly6G+), inflammatory monocytes

(TCRb2, CD192, NK1.12, CD11c2, CD11b+, Ly6G2, Ly6Chi). TCM, central memory; TEM, effector memory. **p , 0.01, ****p , 0.0001. n.s., p . 0.05.

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blot, whereas Q60X and iM1I mutants are absent (Fig. 6D).Analysis of the C-terminal HA tag reveals a number of proteins inall cases. The Q60X and iM1I mutant RAG1 constructs could onlyproduce the smaller isoforms (Fig. 6E).To interrogatewhether thesmaller bands were indeed produced by internal translation, thefirst five internal methionines (AUG) were mutated to isoleucines(AUC) ina sequential and additivemanner (i.e.,M3I =M3I+M2I+M1I). As before, RAG1 constructs were transfected into 293T cellsand only the full-length, ;120-kDa RAG1 isoform is detected viaFLAG probing (Fig. 6F). As hypothesized, the potential TIS at M1,M4, and M5 are required for the expression of their respectivesmaller isoforms as observed by the loss of specific bands uponAUG to AUCmutation (Fig. 6G). Whether the M2 TIS gives riseto the band at;80 kDa is inconclusive, asM2Imutation shows aslight decrease in protein quantity at the putative M2 band butnot complete ablation as with M1, M4, and M5. It is clear thatmutation of the TIS at M3 does not impact expression of theputative M3 isoform. We also interrogated the strength of theKozak sequences surrounding the putative TIS sites using apublished equation (21) and term these “Noderer scores.” Weshow that M1, M2, M4, andM5 all have extremely strong Kozaksequences (Fig.4H).Comparisonof theNodererscores (21)betweenKozak sequences surrounding the putative TIS sites, and sequencessurrounding random, out-of-frame AUGs within the RAG1 tran-script, highlights that these high scores are unlikely to be randomand may be selected for (Fig. 4I). Evolutionary selection for thesealternative TIS sites is supported by conservation of strong internal

TIS across species (Supplemental Fig. 3). Given the degeneratenature of codon specificity, it may not be expected that these Kozaksequences would be selected on the basis of the full-length proteinalone, as synonymous mutations would not impact this, yet theymay impact the natural formation of N-truncated RAG1 isoforms.Overall, these data highlight that RAG1 can be translated frominternal TIS and that these N-truncated RAG1 isoforms may havebeen evolutionarily selected.

DISCUSSION

In this article, we describe a mechanism for the formation ofalternative RAG1 isoforms. These RAG1 isoforms arise as aresult of internal translation initiation and lack N-terminalregions of RAG1. They were revealed upon identification of anovel (to our knowledge) nonsensemutation in RAG1, a geneticlesion that is very similar to that present in a number of OSpatients. We therefore present a novel (to our knowledge)murine model of OS-like disease that describes mechanisti-cally how functional RAG1 protein can be made despite earlynonsense or frameshift mutations and a more-detailed de-scription of T and B cell development in mice with suchmutations. This provides a model to further interrogate howautoimmunity manifests concurrently with immunodeficiencyand the effect that RAG1 protein or defective recombination hasto play in this.

FIGURE 5. RAG1NX mice develop increased age-associated B cells and ANA.

(A–D) T cells from 4- to 5-wk-old WT and NX mice were stimulated ex vivo with PMA and ionomycin, and cytokine expression was measured by

flow cytometry. Statistics: one-way ANOVA with Tukey honest significant difference posttest. Combined data from two independent experiments.

(E) Twenty- to twenty-two–month-old WT and NX mice were compared for peripheral leukocyte populations. Gating: neutrophils (CD32, Ly6G+),

B cells (CD32, Ly6G2, CD19+), T cells (CD3+, CD192, Ly6G2, CD4+, or CD8+), eosinophils (CD32, CD192, Ly6G2, Siglec-F+), macrophages (CD32,

CD192, Ly6G2, Siglec-F2, F4/80+), monocytes (CD32, CD192, Ly6G2, Siglec-F2, F4/802, CD11b+). (F) Frequency of ABCs of total B cells was

quantified in 20- to 22-mo-old female WT and NX mice. Statistics: Mann–Whitney U test. (G) ANA staining intensity on Hep-2a cells was assayed for

in young (4- to 5-wk-old) and old (20- to 22-mo-old) WT and NX mice. Bars indicate mean 6 SEM. Statistics: one-way nonparametric ANOVA with

Tukey honest significant difference posttest. See representative images in Supplemental Fig. 2. *p , 0.05, ***p , 0.0001, ****p , 0.0001.

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The Q60X nonsense mutation in RAG1 would upon initialobservation be expected to result in a nonfunctional peptide thatcould not participate in VDJ recombination, as it should lack thecatalytic domain of RAG1 (the RAG1core domain). However, someinternal translation initiation products provide N-terminal trun-cated RAG1 proteins that contain the RAG1core domain. In fact,many of the phenotypes displayed by the RAG1NXmouse replicatethat of the RAG1core mouse, including blocks in development atAg rearrangement stages, likely because of inefficient recombina-tion activity (6, 22). Interestingly, altered selection of Vb proteinsfrom the DP to SP stage is also seen in the RAG1core mice (6). Thisaltered Vb usage is independent of choice of TCRb rearrangementbut may be due to altered TCRa repertoire or reduced second-ary Va-to-Ja rearrangements. Thus, both in terms of lymphocyte

numbers and VDJ rearrangement, the RAG1NX mouse pheno-copies RAG1core, consistent with the RAG1NX mutation un-expectedly generating smaller N-truncated isoforms.

As a result of decreased recombination efficiency, youngRAG1NX mice expectedly display peripheral B and T celllymphopenia. The increased frequencies of memory phenotypeT cells are likely a result of homeostatic expansion of T cellswithin lymphopenic hosts (23) and increased Treg frequenciesbecause Tregs outcompete conventional T cells (24) or convertfrom conventional T cells in a lymphopenic environment (25).Treg and T memory populations are equivalent in number inyoung mice, and this homeostatic expansion is sufficient to fillthe entire T/B lymphocyte niche upon aging of the mice. Therole of lymphopenia in driving autoimmune disease has been

FIGURE 6. Smaller RAG1 isoforms arise via internal translation initiation.

(A) RAG1 schematic highlighting positions of FLAG and HA epitope tags, Q60X mutation, and internal methionines that serve as potential TIS. (B and

C) Expression of RAG1-WT, Q60X, and iM constructs in 293T cells assayed for FLAG and HA expression by flow cytometry. Pregated on GFP+ cells.

(D and E) Expression of RAG1-WT, Q60X, and iM mutants in 293T cells analyzed by SDS-PAGE and Western blot for FLAG (D) and HA (E) expression.

(F and G) RAG1 WT versus internal methionine mutants (M1–M5) expressed in 293T cells were analyzed by SDS-PAGE and Western blot for FLAG (F)

and HA (G) expression. All blots are representative of at least three independent experiments. (H) Analysis of strength of internal putative TIS Kozak

sequences compared with canonical Kozak sequence and Noderer consensus sequence (21). (I) Noderer scores of putative TIS compared with

Noderer scores surrounding random, out-of-frame AUG sequences within murine RAG1 transcript.

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postulated a number of times, yet it is difficult to tease apart therelative contributions of lymphopenia from altered lymphocytedevelopment, defective VDJ rearrangement, and altered Agreceptor repertoires. It is, however, attractive to speculate thathomeostatic expansion of few T cells drives oligoclonality andthat those T cells that escape development with higher self-reactivity can expand disproportionally.

Although the RAG1NX mouse has a similar genetic lesion tohumans with OS, and similar lymphopenia, the pathology expe-rienced by OS humans is not entirely replicated. However, thereis some evidence of immune dysregulation in that RAG1NX

T cells can more efficiently make effector cytokines ex vivo, andaged RAG1NX mice have an increase in age-associated B cells andincreased ANAs. It is also important to note that, in comparingthe RAG1NX mouse to two other murine models of OS, the MMmouse (15) and the R229Q RAG2 mouse (16), we see a number ofsimilarities and differences. All three models display a block indevelopment of B and T cells at Ag receptor gene rearrangementsteps, increases in memory phenotype T cells, and altered TCRrepertoires. TheMM and R229Q RAG2mice show some facets ofcanonical OS, such as erythroderma and high IgE, but differsubstantially in other aspects, suggesting the specific geneticlesion, mouse strain (which differs between all three mice), andenvironment in which they are housed all may contribute to theobserved autoimmune phenotypes. Of note, the C57BL/6 strainof mice that our RAG1NX mutation is present in is notoriouslyresistant to autoimmunity in the absence of additional environ-mental stimuli (26). Similarly, the R229Q RAG2 mutationon the C57BL/6 background does not present with overtautoimmunity (27).

The most important finding described in this report is that a59 nonsense mutation does not lead to loss of functional RAG1protein. This raises a number of important conceptual points.First, that RAG1 has many naturally occurring, N-truncatedisoforms is of considerable interest. RAG1 has been studied formore than 30 y as a single protein, yet it is intriguing to considerthatnaturally occurring smallerRAG1 isoformsmayhave importantroles innormalRAGfunction.Alreadyweknowthat theNterminusof RAG1 has important functions in aiding recombination activity.For example, the RAG1 N terminus contains an E3 ubiquitin ligasethat modifies histones to enhance RAG cleavage (7, 10) and regionsthatbindtheVprBPkinase tohelprepairRAGDSBs(8).Translationfrom M1 would retain the RAG1 RING domain but may ablateVprBP binding, whereas translation from M2 would likely ablateRINGdomain activity. Translation fromM4andM5would result inloss of portions of the RAG1core domain and will likely have norecombination activity. Whether these internally translated pro-teins are present at biologically significant levels is of considerableinterest because smaller RAG1 isoforms could form part of theRAG12/RAG22 (RAG) heterotetrametric complex and be importantfor the regulation of normal function.

Second, that alternative translation initiation may be a naturalphenomenon occurring widely within eukaryotic cells is still anunderappreciated occurrence and may have many importantconsequences. Ribosome profiling in eukaryotic cells implies

internal translation initiation generates N-truncated isoforms for;15% of the;20,000 human proteins (28–30), yet our understand-ingofmechanisms thatcontrol internal translation initiationandhowresulting N-truncated isoforms function normally and contribute todiseaseareat infancy. Identificationofalternative translationofRAG1contributes to the expanding database of alternatively translatedproteins, and the exactmechanismof translation of RAG1 alternativeisoforms is a subject of future investigation.

Anothermajor consequence of internal translation initiation isthat early nonsense mutations or frameshift mutations leading toearly translation terminationmay not always result in the absenceof a protein. Indeed, it is attractive to speculate that internal TISmay exist as a mechanism by which to protect from deleteriousnonsense or frameshift mutations, as is the case with the RAG1NX

mice that do not have the expected SCID phenotype. This isimportant to consider during analysis of clinical exome/genomesequencing as interpretations could vary drastically. For example,N-terminal truncations of certain proteins may delete autoregu-latory domains and result in constitutively active proteins, ratherthan complete absence. Finally, it is an important factor to considerwhen designing conditional knockout mice using the cre-loxsystem, in which typically exons are flanked by loxP sites andexcised following cre expression, leading to frameshifted proteins.Although the assumption is that this frameshifted mRNA will bedegraded by nonsense-mediated decay, it is possible that trans-lation initiation downstream of the excised region may result inprotein expression. Continued investigation of internal translationinitiation using RAG1 as a model system will provide a means tounderstand all of these possibilities.

DISCLOSURES

The authors have no financial conflicts of interest.

ACKNOWLEDGMENTS

We thank David Schatz (Yale University, New Haven, CT) for providingthe RAG1 Ab and Philip Zoltick (Children’s Hospital of Philadelphia) forthe pZHK expression vector.

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