Bühler Marc, Paillusson Alexandra, Mühlemann Oliver
Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland.
Nucleic Acids Res. 2004 Jun 21;32(11):3304-15. doi: 10.1093/nar/gkh651. Print 2004.
Premature translation-termination codons (PTCs) elicit rapid degradation of the mRNA by a process called nonsense-mediated mRNA decay (NMD). NMD appears to be significantly more efficient for mRNAs of genes belonging to the immunoglobulin superfamily, which frequently acquire PTCs during VDJ rearrangment, than for mRNAs of other genes. To identify determinants for efficient NMD, we developed a minigene system derived from a mouse immunoglobulin micro gene (Ig-micro) and measured the effect of PTCs at different positions on the mRNA level. This revealed that PTCs located downstream of the V-D junction in the VDJ exon of Ig-micro minigenes and of endogenous Ig-micro genes elicit very strong mRNA downregulation, whereas NMD efficiency decreases gradually further upstream in the V segment where a PTC was inserted. Interestingly, two PTCs are in positions where they usually do not trigger NMD (<50 nt from the 3'-most 5' splice site) still resulted in reduced mRNA levels. Using a set of hybrid constructs comprised of Ig-micro and an inefficient substrate for NMD, we identified a 177 nt long element in the V segment that is necessary for efficient downregulation of PTC-containing hybrid transcripts. Moreover, deletion of this NMD-promoting element from the Ig-micro minigene results in loss of strong NMD.
过早的翻译终止密码子（PTCs）会通过一种称为无义介导的mRNA降解（NMD）的过程引发mRNA的快速降解。对于免疫球蛋白超家族基因的mRNA，NMD的效率似乎明显高于其他基因的mRNA，免疫球蛋白超家族基因在VDJ重排过程中经常获得PTCs。为了确定高效NMD的决定因素，我们开发了一种源自小鼠免疫球蛋白微基因（Ig-微基因）的小基因系统，并测量了不同位置的PTCs对mRNA水平的影响。这表明，Ig-微基因小基因和内源性Ig-微基因的VDJ外显子中位于V-D连接下游的PTCs会引发非常强烈的mRNA下调，而在插入PTC的V片段中，NMD效率在更上游逐渐降低。有趣的是，两个PTCs位于通常不会触发NMD的位置（距离最3'端的5'剪接位点<50 nt），但仍导致mRNA水平降低。使用一组由Ig-微基因和低效的NMD底物组成的杂交构建体，我们在V片段中鉴定出一个177 nt长的元件，该元件对于有效下调含PTC的杂交转录本是必需的。此外，从Ig-微基因小基因中删除这个促进NMD的元件会导致强烈NMD的丧失。
