Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, 60800 Multan, Pakistan.
Proc Natl Acad Sci U S A. 2020 Dec 8;117(49):31343-31352. doi: 10.1073/pnas.2004810117. Epub 2020 Nov 23.
Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive allele is expressed, a phenomenon known as allelic exclusion. In contrast to a productively rearranged allele, the messenger RNA (mRNA) () from a nonproductively rearranged allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the mouse model from mice having a premature termination codon at position +5 in leader exon of allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of message, indicating that previous conclusions regarding a role of in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the knock-in allele, which generated stable but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not expression.
祖 B 细胞（ProB 细胞）向前 B 细胞（PreB 细胞）的分化受免疫球蛋白重链检查点（IgHCC）调控，在此过程中，由一个有功能重排的 等位基因编码的免疫球蛋白重链与前 B 细胞受体复合物（PreBCR）组装，产生信号启动这一转变，并抑制抗原受体基因重组，从而确保只有一个有功能的 等位基因被表达，这一现象被称为等位基因排斥。与有功能重排的 等位基因不同，非功能重排的 等位基因的信使 RNA（mRNA）（）会被无意义介导的降解（NMD）所降解。这一事实使得我们无法确定稳定的 等位基因对与 IgHCC 相关的分子和发育变化的贡献。为了解决这一问题，我们构建了一个 等位基因在 5'端前导外显子的+5 位有一个终止密码子的 小鼠模型。这一突变阻止了 NMD 的发生，并且由于跨过早的 Ter5 终止密码子的通读翻译，缺失跨膜区（∆TM）阻止了任何可能形成的信号有效 PreBCR 复合物的形成。一个高度敏感的夹心 Western blot 揭示了 基因的通读翻译，表明之前关于 等位基因在建立等位基因排斥中作用的结论需要进一步探索。通过 RNA 测序（RNA-Seq）确定，这种低水平的 IgHC 足以启动通常与 PreBCR 信号相关的 PreB 细胞标记物的表达。相比之下， 敲入等位基因虽然能够产生稳定的 ，但无法检测到 IgHC，也无法诱导 PreB 细胞的发育。我们的数据表明，IgHCC 是在 IgHC 水平而不是 表达水平上受到调控的。
