Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA.

Abstract

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...

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

“…In both prokaryotes and eukaryotes nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene (Morse and Yanofsky 1969;Losson and Lacroute 1979;Maquat et al 1981;Pelsy and Lacroute 1984;Baumann et al 1985;Nilsson et al 1987;Daar and Maquat 1988;Urlaub et al 1989;Cheng et al 1990;Gozalbo and Hohmann 1990;Barker and Beamon 1991;Gaspar et al 1991;Leeds et al 1991;Baserga and Benz 1992;Lim et al 1992;Cheng and Maquat 1993). Results of this study and others indicate that in S. cerevisiae this nonsense-mediated effect can be attributed to cytoplasmic events that are concurrent with mRNA translation.…”

Triosephosphate isomerase deficiency: Facts and doubts

“…Patients with Glu104Asp/ Arg189Stop and Phe240Leu/Glu145stop compound heterozygosity have also been described (6,7,29). TPI mRNAs with premature stop codons were the first to be shown to be efficiently targeted by nonsense-mediated mRNA decay, dramatically reducing the cytoplasmic half-life of the TPI mRNA (6,7,28). Due to nonsense-mediated mRNA decay, it is not known whether any appreciable truncated proteins are expressed from this class of TPI mutation.…”

The human PAF complex coordinates transcription with events downstream of RNA synthesis

“…This requires RNA surveillance to ensure high fidelity of gene expression. In mammals, nonsense-mediated decay (NMD) occurs not only in the cytoplasm, but also in the nucleus (Humphries et al 1984;Takeshita et al 1984;Cheng and Maquat 1993;Maniatis and Reed 2002). More recently, precursor RNAs containing nonsense codons were found to accumulate near the site of transcription in mammals (Muhlemann et al 2001), suggesting that surveillance occurs around this region.…”