Perturbation of thymocyte development innonsense-mediated decay (NMD)-deficient micePamela A. Frischmeyer-Guerrerioa,1, Robert A. Montgomeryb,1, Daniel S. Warrenb, Sara K. Cookec, Johannes Lutzd,Christopher J. Sonnendayb, Anthony L. Guerrerioa, and Harry C. Dietza,c,2

Departments of aPediatrics and bSurgery, and cHoward Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205; anddDepartment of Internal Medicine III, Nikolaus Fiebiger Center of Molecular Medicine, Friedrich Alexander University Erlangen-Nurnberg, D-91054 Erlangen,Germany

Edited* by Klaus Rajewsky, Immune Disease Institute, Boston, MA, and approved May 3, 2011 (received for review December 22, 2010)

The random nature of T-cell receptor-β (TCR-β) recombinationneeded to generate immunological diversity dictates that two-thirds of alleles will be out-of-frame. Transcripts derived from non-productive rearrangements are cleared by the nonsense-mediatedmRNA decay (NMD) pathway, the process by which cells selectivelydegrade transcripts harboring premature termination codons.Here, we demonstrate that the fetal thymus in transgenic mice thatubiquitously express a dominant-negative form of Rent1/hUpf1, anessential trans-effector of NMD, shows decreased cell number, re-duced CD4CD8 double-positive thymocytes, diminished expressionof TCR-β, and increased expression of CD25, suggesting a defect inpre-TCR signaling. Transgenic fetal thymocytes also demonstrateddiminished endogenous Vβ-to-DβJβ rearrangements, whereas Dβ-to-Jβ rearrangementswere unperturbed, suggesting that inhibitionof NMD induces premature shut-off of TCR-β rearrangement. De-velopmental arrest of thymocytes is prevented by the introductionof a fully rearranged TCR-β transgene that precludes genera-tion of out-of-frame transcripts, suggesting direct mRNA-mediatedtrans-dominant effects. These data document that NMD has beenfunctionally incorporated into developmental programs duringeukaryotic evolution.

RNA | T-cell receptor rearrangement | allelic exclusion | B-cell receptorrearrangement | immune development

Frameshift or nonsense mutations are a common cause ofinherited and acquired genetic diseases, suggesting that

nonsense-mediated decay (NMD) may have evolved to protectthe organism from the deleterious consequences of truncatedproteins that would result if nonsense transcripts were stable.Indeed, NMD modulates the severity of several disease pheno-types (1–3). Recent evidence suggests that the pathway alsoparticipates more broadly in the regulated control of gene ex-pression. NMD has been shown to regulate the stability ofphysiological transcripts that mimic the architecture of nonsensemRNAs in both yeast and mammals (4–6). The nonsense sur-veillance machinery also appears to function in clearing pre-mature termination codon (PTC)-harboring transcripts resultingfrom inefficient, alternative, or faulty RNA processing eventsoccurring during transcription and splicing.The Upf proteins (Upf1p, Upf2p, and Upf3p) have been shown

to be essential trans-effectors of NMD in yeast (7–10). Humanorthologs of each of these proteins have been identified, termedRent1–3/hUpf1–3 (11–15). Complete loss of NMD in lowereukaryotes is well tolerated (16–19). However, homozygous tar-geted disruption of Rent1/Upf1 in mice resulted in stabilizationof nonsense transcripts to WT levels and death at the periim-plantation stage of development (20). Heterozygous targetedmice were completely competent in their ability to perform NMDand were fertile, had normal life spans, and showed no apparentphenotypic abnormalities (20). Recently, using a tissue-specificknock-out approach,Upf2was shown to be essential for survival ofhematopoietic stem and progenitor cells (21).

One process unique to higher eukaryotes where NMD is an-ticipated to be of paramount importance is maturation of theimmune system. During normal lymphocyte development, T-cellreceptor (TCR) and immunoglobin genes undergo a series ofprogrammed gene rearrangements. For TCR-β, this process ini-tiates with joining of a diversity (Dβ) segment to a joining (Jβ)segment. This is followed by a second recombination event inwhich the DβJβ unit is joined to a variable (Vβ) segment. Onemechanism for generating diversity in the T-cell repertoireinvolves the addition and subtraction of nucleotides at the VβDβand DβJβ junctions. Although these events function to increasethe repertoire of TCRs capable of recognizing different antigens,the process is random and, as a result, two of three rearrange-ments result in the production of a frameshift and subsequentPTC (22). In the one-third of cells that have a productive rear-rangement on the first try (β+β0), rearrangement of the secondallele is inhibited through a poorly defined process termed allelicexclusion. The remaining two-thirds of cells (β−β0) rearrange thesecond allele; one-third of these events will be productive (β−β+),and two-thirds will be nonproductive (β−β−). The nonsense tran-scripts derived from out-of-frame TCR-β alleles (β−) are effi-ciently degraded by the NMD pathway (23). The physiologicalimportance of this phenomenon was recently suggested by theabsence of single positive T cells carrying β− alleles in the pe-riphery of mice conditionally deleted for Upf2, suggesting thatcomplete loss of Upf2 is lethal to developing T cells that harbora β− allele (21). Only cells expressing a productively rearrangedTCR-β allele (β+) undergo positive selection and survive.Our understanding of the physiological importance of Rent1/

hUpf1 and NMD in higher eukaryotes has been limited by theembryonic lethality of mice completely lacking function of thepathway. We have therefore generated a transgenic (Tg) mousethat ubiquitously expresses a dominant-negative form of humanRent1/hUpf1 containing a single amino acid change (R844C) inthe highly conserved helicase domain of the protein that waspreviously shown to cause partial stabilization of nonsense tran-scripts in mammalian cells (24). Tg mice were viable and fertilewith no gross phenotypic abnormalities. Here, we show that theydemonstrate a crisis in development of the thymus coincident withthe onset of TCR-β allele rearrangement. The phenotype in-cluded clonal dropout of cell populations, reduced total thymo-cyte cell number, a dramatic paucity of double-positive (DP)thymocytes with a corresponding increase in CD25high double-negative (DN) cells, and reduced expression of TCR-β relative to

Author contributions: P.A.F.-G., R.A.M., D.S.W., and H.C.D. designed research; P.A.F.-G.,R.A.M., D.S.W., S.K.C., J.L., C.J.S., and A.L.G. performed research; P.A.F.-G., R.A.M., D.S.W.,S.K.C., J.L., C.J.S., and A.L.G. analyzed data; and P.A.F.-G. and H.C.D. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1P.A.F.-G. and R.A.M. contributed equally to this work.2To whom correspondence should be addressed. E-mail: hdietz@jhmi.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019352108/-/DCSupplemental.

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WT littermates, suggesting arrest at the pre-TCR stage of de-velopment. These changes could be prevented by introduction ofa fully rearranged TCR-β allele that effectively precludes thegeneration of out-of-frame TCR-β transcripts. Moreover, Tgmicedemonstrated reduced frequency of Vβ-to-DβJβ rearrangements,which are subject to allelic exclusion, but normal frequency ofDβ-to-Jβ rearrangements, which are not. In summation, thesedata suggest that stabilized TCR-β nonsense transcripts may besufficient to inhibit TCR rearrangement and, therefore, thatNMD has been functionally incorporated into critical develop-mental programs during eukaryotic evolution.

ResultsGeneration and Characterization of Rent1/hUpf1 DN Tg Mice. Ahuman Rent1/hUpf1 cDNA encoding a mutant protein (R844C)with documented dominant-negative activity was introduced intofertilized murine oocytes using traditional Tg technology (Fig.S1). The founder that carried the highest copy number of thetransgene was runted, produced no offspring, and died at ∼4 moof age. A Tg littermate with a slightly reduced copy number wasviable and fertile, allowing derivation of the line used for thesestudies (Fig. 1A). Importantly, mice derived from an independentfounder showed concordant results, documenting that the observedeffects relate to transgene expression rather than gene disruption atthe insertion site (Fig. S2). Quantitative RT-PCR analysis reveal-ed comparable levels of endogenous WT and mutant (Tg) Rent1/hUpf1 message in the fetal day (Fd) 16 thymus and all adult tissuesexcept the kidney (Fig. 1B). To determine the transgene’s effect onthe efficiency of NMD, fibroblast cell lines generated from Tg

and control littermates were transiently transfected with WT ornonsense-containing (PTC) forms of a TCR-β minigene construct(25). The normalized steady-state abundance of nonsense TCRmessages was increased approximately twofold in Tg vs. WT celllines (Fig. 1C). The transgene was also bred onto the gusmps

background, which carries a single base-pair deletion in the β-glucuronidase gene that generates a downstream PTC and initi-ates NMD (26). The steady-state abundance of the β-glucuronidasenonsense transcript was increased approximately threefold in thethymus of Tg animals relative to their WT littermates (Fig. 1D).These data document relative inhibition of NMD in Rent1/hUpf1dominant-negative mice.

Disruption of Fetal Thymic Development in Rent1/hUpf1 Tg Mice. Adedicated histopathological analysis did not reveal gross or mi-croscopic abnormalities in any tissues of mice harboring theR844C Tg allele, with the exception of the thymus. We hypothe-sized that the developing thymus would be particularly susceptibleto deleterious consequences of NMD inhibition because of thehigh physiological burden of nonsense transcripts derived from theTCR-β locus. Indeed, we observed reduced numbers of thymocytesin fetal Tg animals relative to their WT littermates (7.8 ± 0.9 × 105

vs. 25.0 ± 3.9 × 105, respectively, at Fd17; P = 0.0008) at a de-velopmental stage coincident with the onset of TCR-β gene rear-rangement at approximately Fd16 in the mouse (27–30).During fetal thymic ontogeny, large numbers of thymocytes pass

through a series of well-defined developmental stages in synchrony.The successive stages of T-cell maturation can be defined by ex-pression of surface markers (Fig. 2A). Flow cytometric analysiswith CD4- and CD8-specific antibodies revealed a reduction of DPthymocytes in Tg animals at Fd16 and Fd17 (Fig. 2B). Themajorityof thymocytes in Tg animals demonstrated impaired transitionfrom CD25high to CD25low and reduced expression of TCR-β(Fig. 2 C and D), similar to findings previously reported in miceincompetent for pre-TCR synthesis or signaling (31–37).During T-cell development, precursor cells choose between αβ

and γδ lineages. The decision to enter either the αβ or γδ pathwayoccurs at the DN stage of thymocyte maturation. We stained Fd16thymocytes using an antibody directed against γδ TCR and ob-served no significant difference in the percentage of γδ-positivecells in Tg animals relative to WT controls (Fig. 2E). Taken to-gether, these data suggest that Rent1/hUpf1 dominant-negativeactivity is associated with a block in αβ lineage development asa consequence of impaired pre-TCR function in fetal thymocytes.No increase in the γδ T-cell population is evident at this de-velopmental stage, suggesting that the immature thymocytes ac-cumulating as a result of Rent1/hUpf1 suppression are not shuntedtoward the γδ lineage.

Reduced Efficiency of NMD Disrupts Fetal Thymic Architecture. His-tological examination of thymic architecture in Rent1/hUpf1 Tgmice revealed distinct zones of cellular dropout that were first seenat Fd17 and were unique to Tg thymi (Fig. 3A). By Fd19, theselesions were more numerous, larger in size, diffusely distributedthroughout the cortex, and associated with intense TUNELstaining at their periphery (Fig. 3B andC). These data suggest thatthere is a population of cells in the Tg thymus that is able to remainviable and expand at very early stages of fetal development butthen undergoes crisis after clonal proliferation in the cortex. Ex-pression of CD4CD8, CD25CD44, and TCR-β in Tg thymocytesapproaches WT levels as cellular dropout progresses in the Tgthymus. In adults, the thymocyte profiles for Tg and WT animalsare nearly indistinguishable (Fig. S3 A–C). No histological differ-ences were observed between adult Tg and WT thymi (Fig. S3D).

Productively Pre-Rearranged TCR Allele Rescues the PhenotypeObserved in Rent1/hUpf1 Tg Animals. Although our data were con-sistent with a deleterious consequence for stabilized TCR-β

Fig. 1. Generation and characterization of R844C Rent1/hUpf1 dominant-negative Tg (TG) mice. (A) Southern blot depicting inheritance of the Rent1/hUpf1 transgene. (B) Expression of the human Rent1/hUpf1 transgene relativeto endogenous mouse (WT) message. The upper band represents endogenousRent1/hUpf1 message, and the lower band is derived from the transgene. (C)Northern blot showing the steady-state abundance of WT or nonsense (PTC)TCR-β message in fibroblasts from Rent1/hUpf1 Tg or WT mice transientlytransfected with the corresponding TCR minigene constructs. Neomycin re-sistance gene mRNA, encoded on the same plasmid, served as a control forloading and transfection efficiency. Neo, neomycin resistance gene. Ratio ofnormalized nonsense-to-WT TCR transcript levels is shown. (D) Northern blotanalysis of endogenous thymus β-glucuronidase mRNA from gusmps/gusmps

mice that did (TG) or did not (WT) carry the Rent1/hUpf1 transgene. Endoge-nous β-actin mRNA served as a loading control. β-gluc, β-glucuronidase.

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nonsense transcripts, a nonspecific toxic effect of NMD in-hibition on developing thymocytes could not be excluded. Toaddress this issue, we introduced a fully rearranged TCR-2Ctransgene (38) that prevents endogenous TCR alleles fromrecombining by allelic exclusion, and thereby precludes non-productive rearrangements and the generation of nonsensetranscripts (39–41). Expression of the TCR-2C transgene com-pletely restored CD4CD8 expression, CD25CD44 expression,and thymic cellularity in fetal Tg mice (Fig. 4 and Fig. S4),suggesting that accumulation of out-of-frame TCR-β transcriptsis either directly or indirectly responsible for the thymic pheno-type observed in Rent1/hUpf1 Tg mice.

Rent1/hUpf1 Transgene Leads to an Overrepresentation of Out-of-Frame TCR-β Alleles and a Reduction in Vβ-to-DβJβ Rearrangementsbut Not Dβ-to-Jβ Rearrangements in Fetal Thymi. To determine therelationship between phenotypic abnormalities in the Rent1/hUpf1 Tg mice and the rearrangement status at the TCR-β locusfurther, we determined the representation of in- and out-of-framealleles in WT and Tg mice using a previously described PCR andsequencing strategy (22). We observed a dramatic overrepresen-tation of β− alleles in the Tg thymus at Fd16, compared with WTlittermates (Table S1; 46% vs. 22%, respectively; P = 0.02). Thisdifference was not seen in adult animals (Table S1; 25% vs. 30%,respectively; P > 0.4). A number of possibilities were considered.First, suppression of Rent1/hUpf1 might somehow cause prolongedsurvival of β−β− cells, leading to relative overrepresentation of β−alleles. However, we observed decreased cell number and in-creased cell death. Second, inhibition of Rent1/hUpf1 might im-pair pre-TCR–mediated proliferation of β+β0 cells, again resultingin apparent enrichment of β− alleles. In contrast to this model, weobserved that the rate of cellular proliferation is identical in WTand Tg thymi (Fig. S5). A final possibility was that suppression ofRent1/hUpf1 perturbs progression of TCR-β allele rearrangementin β−β0 cells. In keeping with this hypothesis, fetal Tg thymocytesdemonstrated a markedly diminished frequency of Vβ-to-DβJβrearrangements (Fig. 5). This differencewasmost striking at Fd16,still present but less pronounced at Fd19, and not apparent in 1-wk-old or adult Tgmice (Fig. 5 and Fig. S6). An equal frequency ofDβ-to-Jβ rearrangements in Tg andWT thymocytes suggested thatthe discrepancy in Vβ-to-DβJβ rearrangements is not simplya consequence of reduced thymic cellularity in Tg animals. Thesedata suggest that inhibition of Rent1/hUpf1 either blocks thymo-cyte development before the onset of Vβ-to-DβJβ rearrangementor that it induces a premature shut-off of TCR-β rearrangement.

Rent1/hUpf1 Transgene Leads to Impaired B-Cell Maturation. LikeTCR-β, Ig heavy chain (HC) genes also undergo a series ofprogrammed gene rearrangements that frequently results inproduction of nonsense transcripts normally degraded by theNMD pathway. These rearrangements occur at the CD19+ c-kit+

pro–B-cell stage. Once a productive HC gene has been generated,the μHC is expressed and triggers the transition from the μHC-

Fig. 2. Abnormal T-cell development in fetal Rent1/hUpf1 dominant-negative Tg (TG) mice. (A) Normal thymocyte maturation. Thymocytes DNfor CD4CD8 can be further subdivided into four discrete stages of differ-entiation defined by expression of CD25 and CD44 as shown. Only cells thatexpress a functional TCR down-regulate CD25, up-regulate CD4 and CD8,and undergo selection. Flow cytometric analysis of thymocytes from fetal(at the indicated Fds) WT or Rent1/hUpf1 Tg mice with antibodies to CD4and CD8 (B), CD25 and CD44 (C), TCR-β (D), and TCR-γδ (E). (Insets) Per-centage of gated cells in each quadrant from a representative litter is givenwithin each panel.

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Fig. 3. Distortion of thymic architecture in Rent1/hUpf1 dominant-negativemice. H&E stain of WT and Tg (TG) thymic lobes from Fd 7 (A) and Fd19 (B)mice. (Magnification: A, 40×; B, 10×.) (C) TUNEL assay for DNA fragmentationon histological sections from Fd19 WT and Tg thymus. (Magnification: 20×.)

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negative pro–B-cell stage to the μHC-positive pre–B-cell stage ofdevelopment. The fetal liver is the major site of B-cell de-velopment during embryonal life. Therefore, we analyzed thelivers of newborn mice and observed an increased proportion ofc-kit+ pro-B cells in the CD19+ B-cell populations of Tg mice

compared with WT littermates (30.8% ± 0.9% vs. 19.8% ± 2.5%,respectively; P ≤ 0.0001; Fig. S7). Of these pro-B cells, slightlyfewer cells produced an intracellular μHC in the Tg mice (8.9% ±1.6% vs. 11.5% ± 1.7%; P = 0.0293), indicating a developmentalimpairment before or during VDJ recombination rather than animpairment in the differentiation of μHC+ pro-B to pre-B cells.As a consequence, Tg mice had fewer surface IgM+ cells (6.3% ±1.6% vs. 9.2% ± 1.3%; P = 0.0046). These data suggest thatdeficiency of NMD impairs early B-cell development coincidentwith the timing of nonsense transcript production. Similar resultsare reported in the accompanying paper by Lutz et al. (42), whichshows impaired B-cell development in mice expressing nonsenseμHC transcripts that cannot be degraded by NMD.

Mice Expressing Truncated TCR-β Protein Do Not Recapitulate AnyAspects of the Rent1/hUpf1 Tg Phenotype. The random additionand subtraction of nucleotides at the VβDβ and DβJβ junc-tions result in the generation of a PTC shortly downstream in theTCR-β message in two of three rearrangements. Normally, thesetranscripts are efficiently degraded by the NMD pathway. How-ever, the truncated proteins these mRNAs encode might be ex-pected to increase in abundance when NMD is disrupted, andtherefore may contribute to the phenotype observed in the Rent1/hUpf1 Tg mice. To test this hypothesis, Tg mice [called leader (L)VDJ] were created that would express a truncated protein en-coded by a construct containing only the L and rearranged VDJexons of TCR-β (Figs. S1 and (S8A). This is representative of thetruncated protein that would be predicted to be expressed froma stabilized nonsense TCR-β message. Mice expressing the LVDJtransgene showed no changes in CD4CD8, CD25CD44, or TCR-βexpression in fetal or adult thymocytes compared with their WTlittermates (Fig. S8 B–D). RT-PCR documented expression ofthe transgene in LVDJ mice at Fd17 (Fig. S8E). Therefore, ex-pression of a stable truncated RNA that is fully translationallycompetent to express truncated TCR-β peptides representative ofthose encoded by nonproductively rearranged TCR-β alleles isinsufficient to recapitulate the phenotype observed in Rent1/hUpf1 Tg mice. These data make it highly unlikely that thephenotype observed in Rent1/hUpf1 mice is a result of truncatedTCR-β proteins that accumulate as a result of disruption of NMD.

DiscussionThe NMD pathway has previously been recognized to play acentral role in degrading out-of-frame TCR-β transcripts (23).However, the consequences of inhibiting this function in vivo arelargely unknown. Initial attempts to address this question by tar-geted silencing of Rent1/hUpf1, an essential trans-effector of thepathway, were noninformative because complete loss of functionwas incompatible with embryonic viability (20). Conditional de-letion ofUpf2 revealed that loss of NMD results in death of T cellsharboring β− but not β+ alleles; however, the underlying mecha-nism responsible for this observation is not known. We have gen-erated Tg mice with reduced efficiency of NMD by overexpressinga dominant-negative form of Rent1/hUpf1. These mice were via-ble, and a subset of fetal thymocytes in Tg animals arrests at a stageof development consistent with a defect in pre-TCR signaling, asevidenced by an increased population of CD4CD8 DN cells thatexpress high levels of CD25 and low levels of TCR-β.There aremany lines of evidence to suggest that stabilized TCR-β

nonsense transcripts contribute to the impaired thymocyte matu-ration and expansion seen in the Rent1/hUpf1 Tg mice. First, ab-normal thymocytes in Rent1/hUpf1 animals arrested just before thepre-TCRstageof development (CD25highCD44negativeCD4CD8DN).This correlates temporally with the onset of TCR-β rearrange-ment, an event known to initiate production of a high physiolog-ical burden of substrates for the NMD pathway (23). Second, itwould be predicted that a nonspecific toxic effect imposed byNMD deficiency would affect all thymocytes equally. In contrast,

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Fig. 4. Rescue of thymocyte development by the productively rearrangedTCR-2C transgene. Flow cytometry of Rent1/hUpf1 Tg (TG) and WT thymo-cytes using CD4 and CD8 antibodies in the presence (+TCR-2C) or absence (noTCR-2C) of the pre-rearranged TCR-2C transgene at Fd17. (Insets) Percentageof gated cells in each quadrant from a representative litter is given withineach panel.

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Fig. 5. Diminished endogenous TCR-β gene rearrangements in Rent1/hUpf1Tg (TG) mice. Southern blot analysis of PCR products derived from amplifi-cation of WT and Rent1/hUpf1 Tg thymocyte DNA from Fd16 or 7-d-old miceusing a sense primer in Vβ 8, 10, or 11 (to detect Vβ-to-DβJβ rearrangements)or upstream of Dβ2 (to detect Dβ-to-Jβ rearrangements) and an antisenseprimer located downstream of Jβ2.7. Only rearranged alleles can be ampli-fied using the Vβ and Jβ2.7 primer pairs, with each specific band corre-sponding to the particular J segment (1–5, 7) involved. Both unrearranged(indicated with arrow) and rearranged segments can be detected with theDβ2 and Jβ2.7 primer set. The blot was hybridized with a probe specific forJβ2.7. A segment of the Cμ gene was amplified as a loading control. Ratios ofrearrangements of Rent1/hUpf1 Tg to WT Vβ-to-DβJβ (normalized for theamount of Dβ-to-Jβ rearrangements) are shown.

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histological examination of fetal Rent1/hUpf1 Tg thymi revealeddiscrete zones of cellular dropout, plausibly representing pop-ulations of β−β0 cells, which have an initial nonproductive rear-rangement resulting in the production of potentially deleteriousout-of-frame messages that are now stable because of inhibition ofNMD. β+β0 cells, which productively rearrange a TCR-β allele onthe first try, and therefore do not express any out-of-frame TCR-βmessage, would be exempt from any deleterious effect of NMDinhibition. Third, we see an increase in the frequency of out-of-frame alleles in fetal Tg thymi despite documentation thatsurviving thymocytes proliferate normally. There is no obviousexplanation if one invokes a generalized and nonspecific toxiceffect of NMD inhibition. Finally, arrest at the pre-TCR stageof development can be rescued by breeding a functionally pre-rearranged TCR-β allele onto the Tg background, presumablyattributable to the absence of TCR-β nonsense transcripts becauserearrangement of both endogenous alleles is inhibited throughallelic exclusion.The mechanisms regulating rearrangement at the TCR-β locus

as well as thymocyte maturation and expansion remain an area ofactive research. We observed that thymocytes from fetal NMD-deficient mice demonstrated a normal frequency of Dβ-to-Jβrearrangements, which are not subject to allelic exclusion, butreduced Vβ-to-DβJβ rearrangements, which are subject to allelicexclusion. Our data therefore suggest that stabilized TCR-βnonsense transcripts that only encode the variable domain, whichcannot support pre-TCR assembly or signaling, may contribute toallelic exclusion. Although we cannot exclude the possibility thatthese effects may be mediated by other functions of the NMDpathway or Rent1/hUpf1 itself, this model is supported by recentobservations that stable but nonproductive μHC transcripts cansuppress VDJ recombination in pro-B cells (42). A prior studyhad failed to observe phenotypic consequence after expression ofa TCR-β message with a physiological frameshift mutation in theDβJβ region that could not derive functional TCR-β protein (43).However, because of the downstream PTC, this message is pre-dicted to be recognized and degraded by the NMD pathway.Recently, Schlimgen et al. (44) reported that initiation of mon-oallelic Vβ-to-DβJβ recombination events in developing thymo-cytes results from stochastic interactions of Tcrb alleles withrepressive nuclear compartments. We propose that stable TCR-βRNA may delay further recombination until the outcome ofrearrangement on the first allele is tested, reconciling the time lagparadox inherent to many prior models. In this view, allelic ex-clusion is a multiphasic process that is initiated by interaction ofTcrb alleles with repressive nuclear compartments, maintainedtransiently by stable TCR-β messages, and consolidated by pre-TCR signaling. Normalization of the thymic phenotype in adultdominant-negative Rent1/hUpf1 Tg animals indicates that thisfunction of NMD may only be operative or discernible in fetalthymocytes undergoing maturation in a synchronous wave.Although many studies have established an essential role for

the mature TCR-β chain and TCR-β signaling in regulatingTCR-β rearrangement and allelic exclusion, this study implicatesa role for TCR-β message and NMD in these events. This modelestablishes a new paradigm that mRNAs can have activities in-

dependent of their contribution to protein production. Althoughwell established for noncoding RNAs, this represents a newconcept for RNAs that also encode protein products. An un-anticipated function of NMD, therefore, may be to safeguard theintended correlation between mRNA and protein functions inthese exceptional circumstances.

Materials and MethodsMice. The Rent1/hUpf1 Tg mice express the mutated (R844C) form of humanRent1/hUpf1 (24) from the β-actin promoter. The LVDJ mice expressa transgene consisting of the L and rearranged VDJ exons of TCR-β from thelck promoter. Details of plasmid construction and genotyping methods canbe found in SI Materials and Methods. Mice heterozygous for the gusmps

mutation (B6.C-H2bml/ByBirgusmps/+) were purchased from the Jackson Lab-oratory. Gusmps and TCR-2C mice were genotyped as previously described(20) and by flow cytometry, respectively. All mice used in this study werebred and maintained under pathogen-free conditions. Experiments wereapproved by the Animal Care and Use Committee.

Cell Lines, Transfection Conditions, RNA Isolation, and Northern Blot Analysis.Transfected fibroblast cell lines established from Fd13.5 Tg and WT embryos,and thymic tissue retrieved from Gusmps mice, were analyzed by Northernblot analysis as described in SI Materials and Methods.

RT-PCR. cDNAs from multiple tissues of Rent1/hUpf1 Tg adult mice, Fd16thymi, and human and mouse fibroblast cell lines were used to amplifyRent1/hUpf1. Amplicons were digested with MboI and analyzed by Southernblotting as described in SI Materials and Methods. A similar approach wasused to evaluate LVDJ transgene expression.

Flow Cytometry. Pregnant females from matings between the two lines of Tganimals (Rent1/hUpf1 and LVDJ) and either C57.BL6 or TCR-2C mice werekilled at various days postcoitum. Single-cell suspensions prepared fromthymi or fetal liver were stained and analyzed as described in SI Materialsand Methods. Multiple mice of each genotype were evaluated.

Histology. Thymi were stained with H&E, and the TUNEL assay was performedper the manufacturer’s instructions (Boehringer Mannheim) as described inSI Materials and Methods.

Frame Assay.A PCR assay utilizing 500 ng of genomic thymocyte DNA isolatedusing the QIAamp DNA mini kit (Qiagen) was performed using Vβ2 and Jβ2.2primers and conditions as described previously (22). PCR products werecloned (TOPO TA 2.1 kit; Invitrogen) and sequenced to determine frame. Atleast 45 unique sequences were evaluated to assess frame.

TCR-β Rearrangement Assay. TCR-β DJ and VDJ gene rearrangements weredetermined by semiquantitative PCR analyses using primers and conditionsas previously described (45).

Statistics. Statistical comparisons were done using the Mann–Whitney Ustatistic, Student t test, or χ2 test (Prism software; GraphPad). P values lessthan 0.05 were considered significant.

ACKNOWLEDGMENTS. We thank Miles Wilkinson for the TCR-minigeneplasmids, Xiaoming Zou for the lck promoter plasmid, Mark Sands for theβ-glucuronidase cDNA, and Jonathon Schneck for the TCR-2C mice. This workwas supported by grants from the National Institutes of Health (to H.C.D.),the Howard Hughes Medical Institute (to H.C.D. and P.A.F.-G.), and theMedical Scientist Training Program (to P.A.F.-G. and A.L.G.).

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