A Quality Control Pathway That Down-regulates Aberrant T-cell Receptor (TCR) Transcripts by a Mechanism Requiring UPF2 and Translation

Abstract

Nonsense-mediated decay (NMD) is an RNA surveillance pathway that degrades mRNAs containing premature termination codons (PTC). T-cell receptor (TCR) and immunoglobulin (Ig) transcripts, which are encoded by genes that very frequently acquire PTCs during lymphoid ontogeny, are down-regulated much more dramatically in response to PTCs than are other known transcripts. Another feature unique to TCR, Ig, and a subset of other mRNAs is that they are down-regulated in response to nonsense codons in the nuclear fraction of cells. This is paradoxical, as the only well recognized entity that recognizes nonsense codons is the cytoplasmic translation apparatus. Therefore, we investigated whether translation is responsible for this nuclear-associated mechanism. We found that the down-regulation of TCR-␤ transcripts in response to nonsense codons requires several features of translation, including an initiator ATG and the ability to scan. We also found that optimal down-regulation depends on a Kozak consensus sequence surrounding the initiator ATG and that it can be initiated by an internal ribosome entry site, neither of which has been demonstrated before for any other PTC-bearing mRNA. At least a portion of this downregulatory response is mediated by the NMD pathway as antisense hUPF2 transcripts increased the levels of PTC-bearing TCR-␤ transcripts in the nuclear fraction of cells. We conclude that a hUPF2-dependent RNA surveillance pathway with translation-like features operating in the nuclear fraction of cells prevents the expression of potentially deleterious truncated proteins encoded by non-productively rearranged TCR genes.

SMG6 interacts with the exon junction complex via two conserved EJC-binding motifs (EBMs) required for nonsense-mediated mRNA decay

“…As reported before, the levels of the PTCcontaining TCR-b and b-globin mRNAs were reduced to ;10% of the wild-type levels (Fig. 1B,D [lanes 8], C; Thermann et al 1998;Wang et al 2002). Depleting SMG6, however, caused the PTC-containing TCR-b and b-globin reporters to increase by sixfold (Fig.…”

“…We monitored NMD using two well-characterized NMD reporters: One was based on a TCR-b minigene (Wang et al 2002), and a second was based on a b-globin gene (Thermann et al 1998). As reported before, the levels of the PTCcontaining TCR-b and b-globin mRNAs were reduced to ;10% of the wild-type levels (Fig.…”

“…Human NMD reporters have been described before (Thermann et al 1998;Wang et al 2002). The plasmid pEGFPC1-NXF1 (Unterholzner and Izaurralde 2004) served as a transfection control.…”

Introns in UTRs: Why we should stop ignoring them

“…Second, NMD clearly serves to dampen the expression of nonfunctional transcripts arising from pseudogenes, expressed transposons, or integrated retroviruses, which frequently contain termination codons upstream of introns [40]. Finally, during programmed gene rearrangements in the T-cell receptor and immunoglobulin genes, unproductively rearranged alleles generate termination codons upstream of introns, and the resulting mRNAs are degraded by NMD [41].…”

SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs

“…Second, STAU1-and UPF2-binding sites within UPF1 overlap so that immunoprecipitation (IP) of STAU1 precludes the detectable co-IP of UPF2 and vice versa. Third, down-regulating STAU1, which inhibits SMD , increases the efficiency of NMD, whereas down-regulating UPF2, which inhibits NMD (Mendell et al 2002;Wang et al 2002;Kim et al 2005;Wittmann et al 2006;Matsuda et al 2007;Woeller et al 2008), increases the efficiency of SMD. Our finding that the differentiation of C2C12 myoblasts (MBs) to myotubes (MTs) is accompanied by an increase in the efficiency of SMD and a decrease in the efficiency of classical (i.e., UPF2 siRNA-sensitive) NMD exemplifies the consequence of competition in light of the fact that the cellular abundance of STAU1 relative to UPF1 decreases less than the cellular abundance of UPF2 relative to UPF1.…”