Eberle Andrea B, Herrmann Kai, Jäck Hans-Martin, Mühlemann Oliver
Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland.
RNA. 2009 Jun;15(6):1021-8. doi: 10.1261/rna.1516409. Epub 2009 Apr 10.
During B cell maturation, immunoglobulin (Ig) genes frequently acquire premature translation-termination codons (PTCs) as a result of the somatic rearrangement of V, D, and J gene segments. However, it is essential for a B lymphocyte to produce only one kind of antibody and therefore to ensure that the heavy and light chain polypeptides are expressed exclusively from the corresponding functional alleles, whereas no protein is made from the nonproductively rearranged alleles. At the post-transcriptional level, a well-studied mRNA quality control mechanism, termed nonsense-mediated mRNA decay (NMD), recognizes and degrades PTC-containing mRNAs in a translation-dependent manner. In addition, transcriptional silencing of PTC-containing Ig-mu and Ig-gamma heavy chain reporter genes was observed in HeLa cells. To investigate the silencing of nonproductively rearranged Ig genes in a more physiological context, we analyzed a monoclonal line of immortalized murine pro-B cells harboring one productively (PTC-) and one nonproductively (PTC+) rearranged Ig-mu heavy chain allele. We show that the steady-state abundance of PTC+ mRNA was approximately 40-fold lower when compared to that of the PTC- mRNA. However, both the PTC+ and PTC- allele seemed to be equally well transcribed since the abundances of PTC+ and PTC- pre-mRNA were very similar and chromatin immunoprecipitations revealed comparable occupancy of RNA polymerase II and acetylated histone H3 on both alleles. Altogether, we found no evidence for transcriptional silencing of the PTC+ allele in this pro-B cell line; hence, the efficient down-regulation of the PTC+ Ig-mu mRNA results entirely from NMD.
在B细胞成熟过程中,由于V、D和J基因片段的体细胞重排,免疫球蛋白(Ig)基因经常获得过早的翻译终止密码子(PTC)。然而,B淋巴细胞仅产生一种抗体至关重要,因此要确保重链和轻链多肽仅从相应的功能等位基因表达,而无义重排的等位基因不产生蛋白质。在转录后水平,一种经过充分研究的mRNA质量控制机制,即无义介导的mRNA降解(NMD),以翻译依赖的方式识别并降解含PTC的mRNA。此外,在HeLa细胞中观察到含PTC的Ig-μ和Ig-γ重链报告基因的转录沉默。为了在更生理的背景下研究无义重排的Ig基因的沉默,我们分析了一个永生化小鼠前B细胞单克隆系,该细胞系含有一个有功能的(PTC-)和一个无义重排的(PTC+)Ig-μ重链等位基因。我们发现,与PTC- mRNA相比,PTC+ mRNA的稳态丰度低约40倍。然而,PTC+和PTC-等位基因的转录似乎同样良好,因为PTC+和PTC-前体mRNA的丰度非常相似,染色质免疫沉淀显示RNA聚合酶II和乙酰化组蛋白H3在两个等位基因上的占据情况相当。总之,我们在这个前B细胞系中没有发现PTC+等位基因转录沉默的证据;因此,PTC+ Ig-μ mRNA的有效下调完全是由NMD导致的。