Mammalian L1 retrotransposon - host interaction

最新发现： ORF 1 p的自适应进化-ORF 1 p是一种RNA分子伴侣，以三聚体形式存在，其单体为三部分，由以下结构组成：功能未知的氨基末端结构域;形成计时体所必需的卷曲螺旋结构域;以及含有高度保守的核酸结合结构域的羧基末端结构域。我们以前表明，ORF 1 p的卷曲螺旋基序经历了人类进化早期的适应性进化。适应性进化通常意味着一个相互作用的系统（例如，病毒及其宿主）。因此，我们使用ORF 1 p作为诱饵在酵母双杂交筛选相互作用的哺乳动物蛋白。经过几轮筛选，我们选择了5个蛋白进行进一步研究：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（hnrnpA 1），DEAD盒多肽（URFH 490），赖氨酰tRNA合成酶。前四个没有与祖先互动（即，ORF 1 p卷曲螺旋结构域的预适应）版本（关于祖先ORF 1 p的更多信息，参见Z 01 DK 057601 -11）。这种相互作用不需要氨基末端结构域，但卷曲螺旋结构域是必需的。所有的宿主蛋白都含有RNA结合基序或已知结合RNA; L1介导的逆转录转座的主要底物。然而，进化分析表明，所有这些宿主蛋白在灵长类动物中高度保守。这一结果表明ORF 1 p的适应性进化并不响应这些宿主蛋白的变化。使用哺乳动物双杂交试验的实验表明，这四种蛋白质，不相互作用与祖先的ORF 1 p在酵母双杂交试验，似乎不太强烈的相互作用与祖先单体比与现代的。我们现在正在确定这种差异是否在宿主蛋白对L1反转录转座的影响中也是明显的。由于所有四种与现代和祖先ORF 1 p差异相互作用的蛋白质都为哺乳动物提供了基本功能，因此它们与L1编码蛋白质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。(See 1 LMCB：1 Z 01 DK 057812 -01哺乳动物L1反转录转座子作为遗传性状。 L1 RNA转运-其他研究表明，核交换因子（NXF 1或Tap 1）结合L1 RNA的3 'UTR区域。NXF 1是介导非剪接RNA从细胞核输出的宿主蛋白，如L1和逆转录病毒RNA的情况。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优先于NXF 1。我们发现，3'非翻译区，据报道，结合NXF 1已高度保守超过60万年的L1进化的灵长类动物。非蛋白质编码序列的强保守性表明它必须为L1复制提供必要的功能。然而，与Marie-Louise Hammarskjld合作，我们无法重现NXF 1与L1 RNA这一区域相互作用的原始报告。我们还在基于细胞培养的测定中测试了该区域的缺失或其他改变对逆转录转座的影响。这些改变没有效果。然而，目前的L1反转录转座测定载体在3 'UTR区域中含有几个主要修饰，这可能妨碍其正常功能。因此，我们正在修改逆转录转座试验，以消除这些变化（见Z 01 DK 057601 -11 LMCB：哺乳动物L1逆转录转座子复制），并重新检查保守区的作用。

RECENT FINDINGS: ADATPIVE EVOLUTION OF ORF1p - ORF1p, which is an RNA chaperone, exists as a trimer whose monomer is tripartite and consists of: an amino terminal domain of unknown function; a coiled-coil domain that is necessary for timer 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. The first four did not interact with the ancestral (i.e., pre-adapted) version of the ORF1p coiled-coil domain (see Z01 DK057601-11, for more information on the ancestral ORF1p). The amino terminal domain is not required for this interaction but the coiled coil domain is essential. All of the host proteins either contain RNA-binding motifs or are known to bind RNA; the primary substrate for L1 mediated retrotransposition. However, evolutionary analysis showed that all of these host proteins were highly conserved in primates. This result suggested that the adaptive evolution of ORF1p was not in response to changes in these host proteins. Experiments using a mammalian two-hybrid assay indicate that the four proteins, which do not interact with ancestral ORF1p in the yeast two-hybrid assay, seem to interact less strongly with ancestral monomers than with the modern ones. We are now determining whether this difference is also evident in the effect of the host proteins on L1 retrotransposition. As all four proteins that interact differentially with modern and ancestral ORF1p serve essential functions for mammals, their putative interaction with L1 encoded proteins could contribute to the genetic load imposed on mammals by L1. (See 1 LMCB: 1 Z01 DK057812-01 Mammalian L1 Retrotransposons as genetic characters.) 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.