Abstract
T-cell receptor-beta (TCRbeta) genes naturally acquire premature termination codons (PTCs) as a result of programmed gene rearrangements. PTC-bearing TCRbeta transcripts are dramatically down-regulated to protect T-cells from the deleterious effects of the truncated proteins that would otherwise be produced. Here we provide evidence that two responses collaborate to elicit this dramatic down-regulation. One is rapid mRNA decay triggered by the nonsense-mediated decay (NMD) RNA surveillance pathway. We demonstrate that this occurs in highly purified nuclei lacking detectable levels of three different cytoplasmic markers, but containing an outer nuclear membrane marker, suggesting that decay occurs either in the nucleoplasm or at the outer nuclear membrane. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCRbeta transcripts escaping to the cytoplasmic fraction of cells. Analysis of TCRbeta mRNA kinetics after either transcriptional repression or induction suggested that this nonsense codon-induced partitioning shift (NIPS) response is not the result of cytoplasmic NMD but instead reflects retention of PTC(+) TCRbeta mRNA in the nuclear fraction of cells. We identified TCRbeta sequences crucial for NIPS but found that NIPS is not exclusively a property of TCRbeta transcripts, and we identified non-TCRbeta sequences that elicit NIPS. RNA interference experiments indicated that NIPS depends on the NMD factors UPF1 and eIF4AIII but not the NMD factor UPF3B. We propose that NIPS collaborates with NMD to retain and degrade a subset of PTC(+) transcripts at the outer nuclear membrane and/or within the nucleoplasm.
Highlights
Generate premature termination codons (PTCs)5 [1]
PTCs Elicit Both Nuclear Fraction mRNA Decay and a Shift in Nuclear-to-Cytoplasmic Fraction mRNA Ratio—We previously demonstrated that PTCs result in the down-regulation of T-cell receptor (TCR) transcripts in the nuclear fraction of mammalian cells [15]
The absence of detectable RCK, calnexin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after the DOC wash indicates that the nuclear fraction preparation has mRNA decay in the nuclear fraction, we examined the half-life negligible levels of cytoplasmic contamination (less than 1%, of PTC-containing (PTCϩ) and PTC-lacking (PTCϪ) TCR based on comparison of the nuclear fraction with dilutions of transcripts in the nuclear and cytoplasmic fractions of HeLa the cytoplasmic fraction)
Summary
We provide evidence that PTCs elicit two responses that collaborate to dramatically reduce the level of PTC-containing TCR transcripts in the cytoplasmic fraction of cells. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCR transcripts escaping to the cytoplasmic fraction of cells. This nonsense codon-induced partitioning shift (NIPS) appeared to not be the result of cytoplasmic NMD, as PTCs did not elicit more rapid cytoplasmic mRNA decay when measured by three independent approaches. Our data suggested that NIPS collaborates with nucleus-associated NMD to dramatically reduce the levels of PTC-bearing transcripts in the cytoplasmic fraction of cells. We propose that NIPS serves as a quality control mechanism that reduces the translation of truncated proteins that would otherwise cause deleterious gain-of-function or dominant-negative effects in mammalian cells
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