Mammalian L1 retrotransposon - host interaction

最新发现： 在L1和哺乳动物生物学中，一个很大程度上未知但重要的问题是L1与其宿主之间的相互作用。我们正在研究两个可能的例子：（1）ORF 1 p的适应性进化ORF 1 p是反转录转座所必需的两个L1编码蛋白之一。ORF 1 p在最近的灵长类历史中经历了适应性进化，这一事件通常意味着生物相互作用（例如，病毒及其宿主）。为了鉴定该事件的可能宿主靶点，我们使用酵母双杂交测定来筛选与目前活跃的人L1 Pa 1家族的适应ORF 1 p相互作用的宿主蛋白，但不与其预适应的祖先版本相互作用，我们从L1 Pa 5家族中复苏。在酵母双杂交试验中的重复测试证实了五种宿主蛋白与ORF 1 p的相互作用，但没有可重复地显示它们与适应和预适应ORF 1 ps的相互作用之间的稳健差异。此外，进化分析表明，5个宿主蛋白是高度保守的，没有经历过适应性反应。然而，哺乳动物双杂交试验表明，一些宿主蛋白强烈干扰多聚体形成的ORF 1 p单体。由于多聚体的形成是ORF 1 p活性所必需的，ORF 1 p和宿主蛋白之间的相互作用可能对宿主和L1都是有害的。我们现在正在确定编码的宿主蛋白是否抑制反转录转座。 (2)L1 RNA转运-其他研究表明，核交换因子（NXF 1）结合L1 RNA的一个区域。NXF 1是一种宿主蛋白质，可以将RNA穿梭进出细胞核。进化分析表明，L1 NXF 1结合位点（部分不编码蛋白质）在灵长类动物的L1进化过程中高度保守。与Marie-Louise Hammarskjld合作的初步实验现在表明，NXF 1确实与L1 RNA结合，我们现在正在确定这种相互作用的结构要求。在我们测试逆转录转座中对NXF 1的需求之前，我们需要修改当前用于测量L1逆转录转座的载体，因为它可能绕过NXF 1介导的RNA转运途径。

RECENT FINDINGS: A largely unknown but major issue in L1 and mammalian biology is the mutual interaction between L1 & its host. We are examining two possible examples: (1) Adaptive evolution of ORF1p ORF1p is one of the two L1 encoded proteins essential for retrotransposition. ORF1p underwent adaptive evolution in recent primate history, an event that often implies a biological interaction (e.g., a virus & its host). To identify possible host targets of this event we used the yeast 2-hybrid assay to screen for host proteins that interact with the adapted ORF1p of the currently active human L1Pa1 family but not with its pre-adapted ancestral version, which we resuscitated from the L1Pa5 family. Repeated testing in the yeast 2-hybrid assay confirmed the interaction of five host proteins with ORF1p but did not reproducibly show a robust difference between their interaction with the adapted and pre-adapted ORF1ps. Also, evolutionary analysis showed that the 5 host proteins were highly conserved & had not undergone an adaptive response. However, a mammalian two-hybrid assay showed that some of the host proteins strongly interfere with multimer formation by ORF1p monomers. As multimer formation is necessary for ORF1p activity, an interaction between ORF1p and the host proteins could potentially be detrimental to both the host and L1. We are now determining whether the encoded host proteins inhibit retrotransposition. (2) L1 RNA transport - Others showed that the nuclear exchange factor (NXF1) binds a region of L1 RNA. NXF1 is a host protein that shuttles RNAs in & out of the nucleus. Evolutionary analysis showed that the L1 NXF1-binding site (part of which does not encode protein) has been highly conserved over 60 Myr of L1-evolution in primates. Preliminary experiments in collaboration with Marie-Louise Hammarskjld now show that NXF1 does indeed bind L1 RNA and we are now determing the structural requirements for this interaction. Before we test the requirement for NXF1 in retrotransposition we need to modify the current vector for measuring L1 retrotransposition as it likely bypasses the NXF1-mediated RNA transport pathway.