The role of 5' splice sites on nuclear mechanisms of regulated gene expression in retroviruses and cellular genes

从DNA模板生成信使RNA是一个复杂的、逐步的过程。mrna获得不同的特征，包括帽状结构和聚a尾部。此外，内含子被剪接出来，建立一个连续的开放阅读框架，用于蛋白质合成。最后，RNA必须通过核孔的活性RNA输出离开细胞核。所有这些步骤的原因之一是“检查点”的实现，以监测mRNA的功能。这些监视机制对于保护细胞免受缺陷rna的侵害至关重要。我们打算研究两个例子，其中细胞质量控制起核心作用。一种是p14基因3'UTR内的点突变，导致一种新的原发性免疫缺陷综合征。这种突变可能产生一个隐式的5 '剪接位点（ss），导致mRNA水平降低。剪接体成分U1 snRNP与p14 mRNA的5 ‘端结合似乎干扰了p14 mRNA正常的3 ’端加工，并可能通过核外泌体导致其快速降解。在第二种情况下，我们计划阐明鼠白血病病毒（MLV）如何调节其含内含子的基因组RNA的输出，MLV是简单γ逆转录病毒的范例。这种RNA上未使用的剪接位点的存在通常会阻止作为另一种质量控制手段的输出。所获得的知识将有助于改进基因转移载体。此外，确定p14免疫缺陷的潜在机制将为了解基因调控提供有价值的见解，并可能为受影响的患者提供治疗策略。

The generation of messenger RNA from a DNA template is a complex, stepwise process. mRNAs acquire different features including a cap-structure and a poly A tail. In addition, introns are spliced out to build a continuous open reading frame for protein synthesis. Finally the RNA has to leave the nucleus via active RNA export through the nuclear pore. One of the reasons for all these steps is the implementation of “check-points” to monitor the functionality of the mRNA. These surveillance mechanisms are essential to protect the cell from defective RNAs. We intend to study two examples where cellular quality control plays a central role. One is a point mutation within the 3’UTR of the p14 gene leading to a novel primary immunodeficiency syndrome. This mutation may create a cryptic 5’ splice site (ss) causing reduced mRNA levels. Binding of the 5’ss by the spliceosomal component U1 snRNP seems to interfere with normal 3’ end processing of the p14 mRNA and leads to rapid degradation, possibly by the nuclear exosome. In the second case, we plan to elucidate how murine leukemia virus (MLV), the paradigm of simple gammaretroviruses, regulates the export of its introncontaining, genomic RNA. The presence of unused splice sites on this RNA would normally prevent export as another means of quality control. The acquired knowledge will contribute to improving gene transfer vectors. Moreover, the identification of the underlying mechanism of the p14 immunodeficiency will provide valuable insights into gene regulation in general, and may also lead to a treatment strategy for the affected patients.