The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to approximately 20% of normal by frameshift and nonsense mutations that cause translation to terminate at a nonsense codon within the first three-fourths of the reading frame. Results of previous studies inhibiting RNA synthesis with actinomycin D suggested that the decrease is not attributable to an increased rate of cytoplasmic mRNA decay. However, the step in TPI RNA metabolism that is altered was not defined, and the use of actinomycin D, in affecting all polymerase II-transcribed genes, could result in artifactual conclusions. In data presented here, the nonsense codon-mediated reduction in the level of TPI mRNA is shown to be characteristic of both nuclear and cytoplasmic fractions of the cell, indicating that the altered metabolic step is nucleus associated. Neither aberrancies in gene transcription nor aberrancies in RNA splicing appear to contribute to the reduction since there were no accompanying changes in the amount of nuclear run-on transcription, the level of any of the six introns in TPI pre-mRNA, or the size of processed mRNA in the nucleus. Deletion of all splice sites that reside downstream of a nonsense codon does not abrogate the reduction, indicating that the reduction takes place independently of the splicing of a downstream intron. Experiments that placed TPI gene expression under the control of the human c-fos promoter, which can be transiently activated by the addition of serum to serum-deprived cells, verified that there is no detectable effect of a nonsense codon on the turnover of cytoplasmic mRNA.The translation of human triosephosphate (TPI) mRNA normally terminates at codon 249 within the final exon, exon 7. In previous experiments that were designed to examine the effect of TPI gene frameshift and nonsense mutations on TPI RNA metabolism, mouse L cells were transiently cotransfected with two plasmids. One plasmid was a pMT-TPI construct, in which the TPI allele harboring the mutation to be tested was driven by the mouse metallothionein (MT)-1 promoter. The other plasmid served to control for variations in cell transfection efficiency, RNA recovery, or both, and was either another pMT-TPI construct or pMT-Gl, in which all but the 5' untranslated region of the TPI allele in pMT-TPI was replaced with the corresponding region of a hybrid human-mouse ,B-globin allele. By using primer extension to quantitate the coexpression of two different MT-TPI alleles or by using Northern (RNA) blot hybridization to quantitate the coexpression an MT-TPI allele and the MT-Gl allele, it was found that nonsense codons that cause translation to terminate prematurely at or upstream of position 189 in the penultimate exon result in a fivefold reduction in the level of MT-TPI mRNA (9, 10). The reduced level was also evident when the MT promoter was replaced by the immediate-early promoter of cytomegalovirus (10). Furthermore, the reduced level was found to be characteristic of both total cell and cytoplasmic RNA but, as evi...

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...

The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations within the TPI gene that cause translation to terminate prematurely at or upstream of codon 189, within exon 6, result in a decreased level of TPI mRNA (I. 0. Daar and L. E. Maquat, Mol. Cell. Biol. 8:802-813, 1988 In animal cells, mRNA abundance is determined by complex nuclear and cytoplasmic processes that include transcription initiation, intron removal, transport from the nucleus to the cytoplasm, and degradation in the cytoplasm. Since mRNA functions primarily to direct the synthesis of protein, it is conceivable that the process of translation could also regulate mRNA abundance. Evidence that this is, in fact, the case exists. As an example, the premature termination of mRNA translation that results from either a nonsense or a frameshift mutation often results in a reduced mRNA level. This has been demonstrated for murine immunoglobulin ,. mRNA (3), human 3-globin mRNA (1,2,16,18,20,23,25,29,31), adenovirus ElA mRNA (13), hamster dihydrofolate reductase (DHFR) mRNA (32), and triosephosphate isomerase (TPI) mRNA (10). For TPI mRNA, in which translation normally terminates at position 249 within exon 7 (the final exon), nonsense codons at position 23 in exon 1, position 69 to 70 in exon 2, position 70 to 71 in exon 2, or position 189 in exon 6 reduce the TPI mRNA level to the same extent, i.e., to 20% of the normal level. One possible explanation for this finding is that sequences between codon 189 (the 3'-most nonsense codon tested) and the normal stop codon at position 249 need to be translated in order to prevent a decrease in the mRNA level. To test this possibility, nonsense codons that reside between positions 189 and 249 were generated and analyzed. Also, deletions were introduced to remove part of exon 6, part of exon 7, or the entire final intron. We conclude from these experiments that nonsense codons in the final exon and, in certain cases, the penultimate exon do not affect the level of TPI mRNA. Therefore, only nonsense codons that reside upstream of the final intron reduce TPI mRNA abundance. We also conclude that this reduction is dependent upon a * Corresponding author.ribosome-free region between the nonsense codon and the final intron. Our data indicate that nonsense codons affect TPI mRNA abundance as a function of their distance upstream of the final intron in TPI pre-mRNA. The effect appears to be limited to newly synthesized RNA, since studies using dactinomycin indicate that the rate of TPI mRNA decay is not altered. MATERIALS AND METHODSDNA-mediated cell transfections and RNA extractions. Murine L tk-cells were transfected by using DEAE-dextran as described previously (10), except that modified Eagle medium containing 10% fetal calf serum and 5% Nu-serum were substituted for Dulbecco modified Eagle medium containing 15% fetal calf serum. The transfecting DNAs consisted of a test plasmid and a reference plasmid. In some experim...

Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination.Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutof of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed.Triosephosphate isomerase (TPI; EC 5.3.1.1) catalyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate and has an important role in glycolysis, gluconeogenesis, fatty acid synthesis, and the pentose shunt (29). Frameshift and nonsense mutations within the human TPI gene that result in a nonsense codon within the first three-fourths of the translated portion of TPI mRNA reduce the abundance of TPI mRNA to 20 to 30% of normal (10,11). Since the reduction is characteristic of both nuclear and cytoplasmic cell fractions and is not attributable to a decrease in the half-life of cytoplasmic TPI mRNA, the degradative process has been rationalized to involve nuclear TPI RNA (10, 11). Such deductive reasoning presents a dilemma when one considers a plausible degradative mechanism because nonsense codons are known to be recognized only in the cytoplasm during the process of mRNA translation-a process that in mammalian cells is generally thought to be spatially and temporally distinct from the processes of nuclear RNA metabolism. Indeed, degradation is likely to be initiated by the premature termination of cytoplasmic mRNA translation, since the reduction in the abundance of a nonsense codoncontaining mRNA is abrogated by a hairpin structure in the 5' untranslated region, which acts in cis to inhibit translation in...

(23,41). In fact, the presence of an intron can influence the way in which at least one nuclear factor associates with the RNA sequences that direct polyadenylation. This was evident with the demonstration that crosslinking of the heterogeneous nuclear ribonucleoprotein C polypeptide to RNA sequences within the SV40 late polyadenylation site is altered by the insertion of an intron, although the functional significance of the alteration is not understood (46). Conversely, mutation of the AAUAAA polyadenylation signal has been shown by using nuclear extracts to depress splicing of the final intron but not the penultimate intron (37). Somewhat at odds with the data that functionally link the final intron to the process of 3'-end formation was the finding that the same chimeric RNA that was used in the Niwa et al. (39) study could be cleaved and polyadenylated after it had been spliced, provided that it was first extracted from agarose and deproteinized (36). The kinetics of cleavage and polyadenylation were similar to those for the unspliced species and, therefore, contraindicated a role of the final intron in 3'-end formation. We rationalized that studies using intact cells so that all cellular factors would be present at physiological concentrations should help to resolve the function of the final intron in 3'-end formation. If a function were indicated, then it would also be possible to localize the relevant intron sequences by deletion analysis.In this report, we begin to examine in cultured cells the 3359 Vol. 13,No. 6 on April 30, 2019 by guest