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, 108 (26), 10638-43

Perturbation of Thymocyte Development in Nonsense-Mediated Decay (NMD)-deficient Mice

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Perturbation of Thymocyte Development in Nonsense-Mediated Decay (NMD)-deficient Mice

Pamela A Frischmeyer-Guerrerio et al. Proc Natl Acad Sci U S A.

Abstract

The random nature of T-cell receptor-β (TCR-β) recombination needed to generate immunological diversity dictates that two-thirds of alleles will be out-of-frame. Transcripts derived from nonproductive rearrangements are cleared by the nonsense-mediated mRNA decay (NMD) pathway, the process by which cells selectively degrade transcripts harboring premature termination codons. Here, we demonstrate that the fetal thymus in transgenic mice that ubiquitously express a dominant-negative form of Rent1/hUpf1, an essential trans-effector of NMD, shows decreased cell number, reduced CD4CD8 double-positive thymocytes, diminished expression of TCR-β, and increased expression of CD25, suggesting a defect in pre-TCR signaling. Transgenic fetal thymocytes also demonstrated diminished endogenous Vβ-to-DβJβ rearrangements, whereas Dβ-to-Jβ rearrangements were unperturbed, suggesting that inhibition of NMD induces premature shut-off of TCR-β rearrangement. Developmental arrest of thymocytes is prevented by the introduction of a fully rearranged TCR-β transgene that precludes generation of out-of-frame transcripts, suggesting direct mRNA-mediated trans-dominant effects. These data document that NMD has been functionally incorporated into developmental programs during eukaryotic evolution.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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 relative to endogenous mouse (WT) message. The upper band represents endogenous Rent1/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 transiently transfected with the corresponding TCR minigene constructs. Neomycin resistance gene mRNA, encoded on the same plasmid, served as a control for loading and transfection efficiency. Neo, neomycin resistance gene. Ratio of normalized nonsense-to-WT TCR transcript levels is shown. (D) Northern blot analysis of endogenous thymus β-glucuronidase mRNA from gusmps/gusmps mice that did (TG) or did not (WT) carry the Rent1/hUpf1 transgene. Endogenous β-actin mRNA served as a loading control. β-gluc, β-glucuronidase.
Fig. 2.
Fig. 2.
Abnormal T-cell development in fetal Rent1/hUpf1 dominant-negative Tg (TG) mice. (A) Normal thymocyte maturation. Thymocytes DN for CD4CD8 can be further subdivided into four discrete stages of differentiation defined by expression of CD25 and CD44 as shown. Only cells that express 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 CD4 and CD8 (B), CD25 and CD44 (C), TCR-β (D), and TCR-γδ (E). (Insets) Percentage of gated cells in each quadrant from a representative litter is given within each panel.
Fig. 3.
Fig. 3.
Distortion of thymic architecture in Rent1/hUpf1 dominant-negative mice. 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 fragmentation on histological sections from Fd19 WT and Tg thymus. (Magnification: 20×.)
Fig. 4.
Fig. 4.
Rescue of thymocyte development by the productively rearranged TCR-2C transgene. Flow cytometry of Rent1/hUpf1 Tg (TG) and WT thymocytes using CD4 and CD8 antibodies in the presence (+TCR-2C) or absence (no TCR-2C) of the pre-rearranged TCR-2C transgene at Fd17. (Insets) Percentage of gated cells in each quadrant from a representative litter is given within each panel.
Fig. 5.
Fig. 5.
Diminished endogenous TCR-β gene rearrangements in Rent1/hUpf1 Tg (TG) mice. Southern blot analysis of PCR products derived from amplification of WT and Rent1/hUpf1 Tg thymocyte DNA from Fd16 or 7-d-old mice using 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 antisense primer located downstream of Jβ2.7. Only rearranged alleles can be amplified using the Vβ and Jβ2.7 primer pairs, with each specific band corresponding to the particular J segment (–5, 7) involved. Both unrearranged (indicated with arrow) and rearranged segments can be detected with the Dβ2 and Jβ2.7 primer set. The blot was hybridized with a probe specific for Jβ2.7. A segment of the Cμ gene was amplified as a loading control. Ratios of rearrangements of Rent1/hUpf1 Tg to WT Vβ-to-DβJβ (normalized for the amount of Dβ-to-Jβ rearrangements) are shown.

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