Mammalian L1 retrotransposon - host interaction

R最近的调查结果： ORF1p-ORF1p是一种RNA伴侣，它的进化是以多聚体形式存在的，其单体是三聚体。它包括：功能未知的氨基末端结构域；形成多聚体所必需的卷曲结构域；以及含有高度保守的核酸结合域的羧基末端结构域。我们之前已经证明，ORF1p的螺旋螺旋基序在古人类进化的早期经历了几次适应性进化。适应性进化通常意味着一个相互作用的系统(例如，病毒和它的宿主)。因此，我们使用ORF1p作为诱饵，在酵母双杂交筛选中与哺乳动物蛋白相互作用。经过几轮筛选，我们选择了5个蛋白质进行进一步的研究：支架附着因子A(SafA)、冷诱导RNA结合蛋白(CIRBP)、异质核RNA结合蛋白A1(HnRNPA1)、死盒多肽(URFH490)和赖氨酰tRNA合成酶。这些蛋白质含有RNA结合基序或已知结合RNA，RNA是L1逆转录转座的主要底物。使用酵母和哺乳动物双杂交分析的实验表明，这些蛋白质与ORF1p的祖先(即预先适应的)和现代(即适应的)版本相互作用不同。(有关祖先ORF1p的更多信息，请参见Z01 DK057601-11)。然而，进化分析表明，所有这些宿主蛋白在灵长类动物中都高度保守。因此，ORF1p的适应性进化显然不是对这些宿主蛋白变化的反应。我们现在正在确定这些宿主蛋白是否对编码现代(适应的)或祖先(前适应的)ORF1p的L1元件促进的L1逆转座子产生不同的影响。由于被测试的宿主蛋白对哺乳动物具有必要的功能，它们与L1编码蛋白的假定相互作用可能有助于L1对哺乳动物施加的遗传负荷。 L1RNA转运-其他研究表明，核交换因子(Nxf1或TAP1)与L1RNA3‘UTR区的一个区域结合。Nxf1是一种宿主蛋白，它介导非剪接RNA从细胞核输出，就像L1和逆转录病毒RNA一样。在哺乳动物中，L1元件相对于其他反转录转座子的优势可能是由于L1先发制人Nxf1。我们发现，据报道与Nxf1结合的3‘非编码区在灵长类L1进化的60Myr以上高度保守。非蛋白质编码序列的高度保守性表明，它必须为L1复制提供必要的功能。然而，在与Marie-Louise Hammarskjeld的合作中，我们无法复制Nxf1与L1RNA的这一区域相互作用的原始报告。我们还在基于细胞培养的实验中测试了该区域的缺失或其他改变对逆转录转座的影响。这些改动没有任何效果。然而，目前的L1逆转录转座实验载体在3‘非编码区包含了几个主要的修饰，这可能会排除其正常的功能。因此，我们正在修改逆转录转座试验，以消除这些变化(参见Z01 DK057601-11 LMCB：哺乳动物L1反转录转座子复制)，并重新检查保守区的作用。

RRECENT FINDINGS: ADATPIVE EVOLUTION OF ORF1p - ORF1p, which is an RNA chaperone, exists as a multimer, the monomer of which is tripartite. It consists of: an amino terminal domain of unknown function; a coiled-coil domain that is necessary for multimer formation; and a carboxyl terminal domain that contains a highly conserved nucleic acid binding domain. We previously showed that the coiled coil motif of ORF1p underwent episodes of adaptive evolution early in hominid evolution. Adaptive evolution often implies an interacting system (e.g., a virus & its host). Therefore, we used ORF1p as bait in a yeast two-hybrid screen for interacting mammalian proteins. After several rounds of screening we chose 5 proteins for further study: scaffold attachment factor A (safA), cold inducible RNA binding protein (CIRBP), heterogeneous nuclear RNA binding protein A1 (hnrnpA1), DEAD box polypeptide (URFH490), and lysyl tRNA synthetase. These proteins contain RNA-binding motifs or are known to bind RNA, the primary substrate for L1 retrotransposition. Experiments using both yeast and mammalian two-hybrid assays showed that these proteins interact differently with the ancestral (i.e., pre-adapted) and modern (i.e., adapted) versions of ORF1p. (See Z01 DK057601-11, for more information on the ancestral ORF1p). However, evolutionary analysis showed that all of these host proteins were highly conserved in primates. Thus, the adaptive evolution of ORF1p was apparently not a response to changes in these host proteins. We are now determining whether these host proteins differentially affect L1 retrotransposition promoted by L1 elements that encode either the modern (adapted) or ancestral (pre-adapted) ORF1p. As the tested host proteins serve essential functions for mammals, their putative interaction with L1 encoded proteins could contribute to the genetic load imposed on mammals by L1. L1 RNA TRANSPORT - Others showed that the nuclear exchange factor (NXF1, or Tap1) binds a region of the 3'UTR of L1 RNA. NXF1 is a host protein that mediates the export of non-spliced RNAs from the nucleus, as would be the case for L1 and retroviral RNAs. The dominance of L1 elements over other retrotransposons in mammals could result from L1 pre-empting NXF1. We found that the 3' UTR region that was reported to bind NXF1 has been highly conserved over 60 Myr of L1 evolution in primates. The strong conservation of a non-protein encoding sequence indicates that it must serve an essential function for L1 replication. However, in collaboration with Marie-Louise Hammarskjld we could not reproduce the original report of NXF1 interaction with this region of L1 RNA. We also tested the effect of deletions or other alterations of this region on retrotransposition in a cell culture based assay. These alterations had no effect. However, the current L1 retrotransposition assay vector contains several major modifications in the region of the 3'UTR, which may preclude its normal function. Therefore we are modifying the retrotransposition assay to eliminate these changes (see Z01 DK057601-11 LMCB: Mammalian L1 retrotransposon replication) and reexamine the role of the conserved region.