Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA.

Abstract

The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to 20-30% of normal by frameshift and nonsense mutations that prematurely terminate translation within the first three-quarters of the reading frame. The decrease has been shown to be attributable to a reduced level of TPI mRNA that copurifies with nuclei. Given that the translational reading frame of an mRNA is assessed in the cytoplasm during protein synthesis, cytoplasmic and nuclear RNA processes may be linked. Alternatively, a nuclear mechanism may exist whereby in-frame nonsense codons can be identified. To differentiate between these two possibilities, two distinct modulators of protein synthesis have been tested for the ability to influence the nonsense-codonmediated reduction in the mRNA level. (s) A suppressor tRNA, which acts in trans to suppress an amber nonsense codon within TPI mRNA, and (it) a hairpin structure in the 5' untranslated region of TPI mRNA, which acts exclusively in cis to inhibit initiation of TPI mRNA translation, were found, individually, and to a greater extent, together, to abrogate the decrease in mRNA. These results show that tRNA and ribosomes coordinately mediate the effect of a nonsense codon on the level of newly synthesized TPI mRNA. We suggest that the premature termination of TPI mRNA translation in the cytoplasm can reduce the level of TPI mRNA that fractionates with nuclei.In eukaryotes, mRNAs that harbor a frameshift or a nonsense mutation and that, as a consequence, prematurely terminate translation are often abnormally low in abundance. Because nonsense codons are recognized in the cytoplasm during protein synthesis, it was rationalized that the premature arrest of translating ribosomes could result in degradation of the template mRNA. The implication that degradation occurs in the cytoplasm appears true in some instances but not in others. For mRNAs that encode yeast URA3 (1) and HIS4(2), Rous sarcoma virus gag (3), rabbit P globin (4), and human 83-globin when the mRNA derives either from the endogenous gene in bone-marrow cells (5) or from a transgene in mice (6, 7), the kinetics of decay during an experimentally induced block in transcription are consistent with the decay process occurring in the cytoplasm and targeting all mRNA molecules that harbor the nonsense codon. However, for the mRNAs that encode human triosephosphate isomerase (EC 5.3.1.1) (TPI; refs. 8, 9, and 26), hamster dihydrofolate reductase (10), nonstructural protein NS2 of the minute virus of mice (11), and human P-globin when the mRNA derives from a simian virus 40-based vector in Syrian hamster cells (12), the decay process does not appear to occur in the cytoplasm and does not affect all mRNA molecules that harbor the nonsense codon. The reduction in abundance of TPI mRNA is characteristic of both nuclear and cytoplasmic fractions, suggesting that the decay process occurs in association with the nucleus (26). TPI gene transcription and RNA splicing appear unaffected by a nonsense codon, and the 20-30o of...

A Regulatory Mechanism That Detects Premature Nonsense Codons in T-cell Receptor Transcripts in Vivo Is Reversed by Protein Synthesis Inhibitors in Vitro

“…5C) is consistent with this possibility. This hypothesis is also supported by a study that showed that ptc-mediated mRNA decay is partially inhibited by the presence of a stable hairpin loop in the 5Ј end of an mRNA or by co-transfection with a nonsense suppressor tRNA (18). Our observation that protein synthesis inhibitors that act by different mechanisms all efficiently reverse the down-regulation of ptc-bearing TCR-␤ transcripts (Fig.…”

Context Effects on Misreading and Suppression at UAG Codons in Human Cells

“…Nevertheless, as long as the 3Ј codon context does not affect mRNA stability through a completely different mechanism, the pattern of context effects we have identified will remain an attribute of the mammalian translational machinery. While it might be expected that the absence of ribosomes from the downstream portion of a message punctuated by a termination codon would render it sensitive to endonucleolytic attack, investigations have indicated that nuclear abundance and the processing of introns can also be altered (4,5,17). In this regard we note that the pRSVgal construct used in our studies contains no introns but does contain a poly(A) addition site (26).…”

Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression

“…In the case of BR mRNA, the translocation of BR through the nuclear pore has been observed by electron microscopy and led to the following conclusions: (1) the 59 end of the mRNA is exported first (Visa et al+, 1996), (2) the protein cover of the mRNA is significantly reduced in the cytoplasm, and (3) translation can take place on a partly translocated mRNA (Daneholt, 1997)+ The complexity of the translocation process for BR and the single orientation of BR mRNA observed during its passage through the nuclear pore complex suggest that, for BR, transport is predominantly or exclusively unidirectional+ The extreme size of BR mRNA (30 kb), as well as the particular ultrastructure of polytene cell nuclei make it difficult to assess the generality of this observation+ Yet, the fact that the nuclear import of large nucleic acid molecules can take place is confirmed by DNA transfection experiments or by the life cycle of influenza virus during which the viral nucleoprotein NP mediates the import of genomic RNA to the nucleus via a classical NLS pathway (O'Neill et al+, 1995)+ A current view of mRNA export is that it is controlled by the protein cover of the transcript and that a mature mRNA could carry a multiplicity of signals that could promote export or retention within the nucleus+ The fact that the localization signals of several hnRNP proteins, like hnRNP A1 and hnRNP K, are functional for both export and import suggests a possible pathway for a retrograde transport of mRNA (Michael, 2000)+ Such a dynamic equilibrium between nuclear and cytoplasmic compartments could create the conditions for a retrocontrol of gene expression by cellular mRNA+ If specific mRNAs could reach back to their site of transcription, they could play a regulatory role, as has been postulated for snRNAs (Frey et al+, 1999)+ More generally, the presence within the nucleus of an image of the cytoplasmic mRNA compartment could constitute a sensing mechanism of cellular metabolism and be involved in a broad transcriptional regulation+ A bidirectional transport would also shed a new light on the mechanism of nonsense-mediated mRNA decay (NMD), which seems to involve a coordination between nuclear and cytoplasmic mRNA metabolism+ On one side, the presence of a premature termination codon (PTC) in an open reading frame causes destabilization of the mRNA, provided that the PTC is located upstream of a functional intron+ Yet, in most cases, destabilization occurs after splicing, as the abundance of pre-mRNA is not modified and the PTC can be recognized as such when overlapping an exon-exon junction (Carter et al+, 1996)+ On the other side, destabilization is intimately linked to translation, as it is abolished in the presence of suppressor tRNA or translation inhibiting 59 stem-loop structure (Belgrader et al+, 1993)+ Recently, a model has been proposed to account for the intron and translation dependence of NMD, based on the possibility that following splicing, proteins may remain bound to the exon-exon junction+ During translation the presence of such a signature downstream of a stop codon could induce the degradation of the mRNA (Thermann et al+, 1998)+ The remaining issue is that, in several instances, mRNA abundance is decreased not only in the cytoplasm but also in the nucleus+ A bidirectional transport would explain this coordinated degradation which otherwise would necessitate the presence in the nucleus of a translation apparatus able to detect nonsense codons+…”