Boundary-independent polar nonsense-mediated decay

Abstract

Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that degrades mRNAs containing premature termination (nonsense) codons. The second signal for this pathway in mammalian cells is an intron that must be at least approximately 55 nucleotides downstream of the nonsense codon. Although the functional significance of this '-55 boundary rule' is not known, it is widely thought to reflect the important role of an exon junction protein complex deposited just upstream of exon-exon junctions after RNA splicing. Here we report that a T-cell receptor (TCR)-beta gene did not conform to this rule. Rather than a definitive boundary position, nonsense codons had a polar effect, such that nonsense codons distant from the terminal downstream intron triggered robust NMD and proximal nonsense codons caused modest NMD. We identified a region of the TCR-beta gene that conferred this boundary-independent polar expression pattern on a heterologous gene. Collectively, our results suggest that TCR-beta transcripts contain one or more sequence elements that elicit an unusual NMD response triggered by a novel second signal that ultimately causes boundary-independent polar regulation. TCR genes may have evolved this unique NMD response because they frequently acquire nonsense codons during normal development.

Fig. 1. Boundary-independent polar NMD regulation. The upper panel shows the positions of nonsense and missense mutations introduced in the Cβ_2.3 exon [the numbers refer to the distance (in nt) between the end of the mutated codon and the exon–intron boundary; the size of the Cβ_2.3 exon is 107 nt]. The lower panel shows RPA of total cellular RNA (10 µg) from HeLa cells transiently transfected with the constructs indicated. The TCR-β mRNA band protected by the TCR-β Vβ probe (position shown) is ∼72 nt, which is the expected size based on the positions of the splice sites in TCR-β mRNA. TCR-β mRNA levels were determined by normalizing against the levels of neomycin (neo) mRNA, which was expressed from the same plasmids as was TCR-β mRNA. Similar results were obtained for each construct in at least three independent transfection experiments.

Fig. 2. Boundary-independent polar NMD regulation is a conserved feature of TCR-β introns. (A) The position of nonsense and missense mutations introduced in the VDJ exon (its total length is 354 nt). (B and C) RPA performed and quantitated as in Figure 1. (D) The effect of premature termination codon (PTC) position on the degree of NMD (PTC^–/PTC⁺ mRNA ratio), based on at least three experiments for each construct, some of which are shown in (B) and (C); error bars indicate standard deviations.

Fig. 3. Boundary-independent polar NMD regulation is independent of nonsense codon context. The upper panel shows the deletions introduced in the VDJ exon. The lower panel shows RPA performed and quantitated as in Figure 1. Similar results were obtained in at least two independent transfection experiments.

Fig. 4. The polar effect is dictated by the distance to the 3′-most intron. The upper panel shows the positions of nonsense and missense mutations introduced in the VDJ exon. The lower panel shows RPA performed and quantitated as in Figure 1. Similar results were obtained in at least three independent transfection experiments.

Fig. 5. The TCR Vβ_8.1Dβ₂Jβ_2.3 exon and adjacent intron sequences are necessary and sufficient for boundary-independent NMD regulation. (A) The TCR-β VDJ segment triggers boundary-independent NMD. Shown are TCR/TPI chimeric transcripts detected with the Vβ probe described in Figure 1. (B) A control segment of similar size (Pem exon 4 and adjacent intron sequences) does not confer boundary-independent NMD regulation. Shown are Pem/TPI (left panel) and TCR/TPI (right panel) chimeric transcripts detected with the TPI probe shown. (C) Deletion of the TCR-β VDJ region prevents boundary-independent NMD regulation. Shown are TCR-β transcripts detected with the Cβ probe shown. RPA was performed and quantitated as in Figure 1. The TPI mRNA band protected by the TPI probe (position shown) is ∼400 nt. The TCR-β mRNA band protected by the TCR-β Cβ probe (position shown) is ∼60 nt, which is the expected size based on the positions of the splice sites in TCR-β mRNA. Similar results were obtained in at least two independent transfection experiments.