Zhang J, Maquat L E
Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
RNA. 1996 Mar;2(3):235-43.
For most of the mammalian mRNAs that have been shown to be reduced in abundance by a nonsense or a frameshift mutation that generates a nonsense codon, reduction takes place while the mRNA is nucleus-associated rather than after the mRNA has been exported to the cytoplasm (reviewed in Maquat LE, 1995, RNA 1:453-465). A variety of mechanisms have been put forth to explain how a nonsense codon could affect the abundance of nuclear mRNA. Some mechanisms have implicated nonsense codon recognition in the nucleus prior to splicing. Among the best-studied nonsense transcripts that manifest nonsense-mediated alterations in nucleus-associated metabolism are those that derive from human alleles for the glycolytic enzyme triosephosphate isomerase (TPI). Nonsense codons within TPI transcripts have been shown to reduce the half-life of completely spliced TPI (mRNA that co-purifies with nuclei (Belgrader P et al., 1994, Mol Cell Biol 14:8219-8228). However, whether or not nonsense codon recognition within TPI transcripts takes place prior to or after splicing remained unresolved. To address this issue, codons that span two exons, i.e., are disrupted by an intron prior to pre-mRNA splicing, were converted to nonsense. If nonsense codon recognition were to precede splicing, then the disrupting intron would be expected to preclude nonsense codon recognition by preventing the physical juxtapositioning of the codon nucleotides. In the absence of nonsense codon recognition, there would be no nonsense-mediated reduction in TPI mRNA abundance. The results of northern (RNA) blot hybridization demonstrated that the two nonsense codons of this type that were studied reduced the level of total, nuclear and cytoplasmic TPI mRNA to an average of 12% of normal, consistent with each nonsense codon being competent to mediate nuclear mRNA degradation. The possibility that the nonsense codons reduced TPI mRNA abundance by altering TPI mRNA abundance or splicing was eliminated by using RT-PCR to demonstrate that the level of each intron within pre-mRNA was essentially unaffected and cDNA sequencing to demonstrate that splice site choice was unaltered. Furthermore, missense codons that harbored some of the nonsense codon changes were found to have little effect on mRNA abundance. These findings, plus the previous finding that a suppressor tRNA abrogates the decay of TPI mRNA brought about by a nonsense codon residing within a single exon (Belgrader P, Cheng J, Maquat LE, 1993, Proc Natl Acad Sci USA 90:482-486), argue strongly that nonsense codon recognition in the nonsense-mediated decay of TPI mRNA takes place after splicing.
对于大多数已被证明因产生无义密码子的无义突变或移码突变而丰度降低的哺乳动物mRNA，其丰度降低发生在mRNA与细胞核相关联时，而非在mRNA输出到细胞质之后（见Maquat LE的综述，1995年，《RNA》1:453 - 465）。人们提出了多种机制来解释无义密码子如何影响核mRNA的丰度。一些机制涉及在剪接之前细胞核中对无义密码子的识别。在研究得最深入的无义转录本中，那些在与细胞核相关的代谢中表现出无义介导改变的转录本，是那些源自糖酵解酶磷酸丙糖异构酶（TPI）的人类等位基因的转录本。已表明TPI转录本中的无义密码子会降低完全剪接的TPI（与细胞核共纯化的mRNA）的半衰期（Belgrader P等人，1994年，《分子细胞生物学》14:8219 - 8228）。然而，TPI转录本中无义密码子的识别是在剪接之前还是之后发生仍未解决。为了解决这个问题，将跨越两个外显子（即在mRNA前体剪接之前被内含子打断）的密码子转换为无义密码子。如果无义密码子的识别先于剪接，那么打断内含子预计会通过阻止密码子核苷酸的物理并列来排除无义密码子的识别。在没有无义密码子识别的情况下，TPI mRNA丰度不会有无义介导的降低。Northern（RNA）印迹杂交结果表明，所研究的这种类型的两个无义密码子将总TPI mRNA、核TPI mRNA和细胞质TPI mRNA的水平平均降低到正常水平的12%，这与每个无义密码子都能够介导核mRNA降解一致。通过使用逆转录 - 聚合酶链反应（RT - PCR）证明前体mRNA中每个内含子的水平基本未受影响，以及通过cDNA测序证明剪接位点选择未改变，排除了无义密码子通过改变TPI mRNA丰度或剪接来降低TPI mRNA丰度的可能性。此外，发现带有一些无义密码子变化的错义密码子对mRNA丰度影响很小。这些发现，加上之前的发现，即一种抑制性tRNA消除了由单个外显子中的无义密码子引起的TPI mRNA的降解（Belgrader P、Cheng J、Maquat LE，1993年，《美国国家科学院院刊》90:482 - 486），有力地表明TPI mRNA无义介导降解中的无义密码子识别发生在剪接之后。
