Reverse Transcriptase Activity in Megakaryocytes and Platelets

描述(申请人提供)：细胞核控制表征单个细胞功能的基因表达模式。发生在细胞核内的反应，如Pre-mRNA的转录和剪接，是利用许多因素的复杂过程。由于血小板在没有核的情况下循环，它们被刻板地认为是一种缺乏复杂性和缺乏核功能的细胞。然而，我们的团队最近发现，血小板携带一个功能性剪接体，并对激活信号做出反应，将前-mRNA拼接成成熟的、可翻译的消息。这一结果是出乎意料的，因为之前人们认为前mRNA剪接只存在于细胞核中。拥有功能剪接体显示了显著的特化和复杂的信息传递，从父母的巨核细胞到终末分化的血小板。这也增加了巨核细胞在血小板生成过程中赋予血小板其他新的基因表达途径的可能性。最近的证据表明，长分布的核元件(LINE-1；也被称为L1元件)反转录转座子家族具有逆转录酶活性，调节细胞的生长、分化和基因表达。这表明L1元素可能调节巨核细胞的生成和原血小板的形成。如果转移到前血小板，L1元件也可能控制成熟、循环中的血小板的功能反应。然而，L1元素是否控制巨核细胞和血小板功能还没有得到测试。因此，在R21的应用中，我们将探索巨核细胞和血小板具有功能L1元件的可能性。在特定的目标1中，我们将描述巨核细胞中的L1机制。我们将确定人类干细胞来源的巨核细胞中是否存在L1编码蛋白，以及它们是否调节巨核细胞的分化和原血小板的形成。在目标2中，我们将描述L1机制在成熟的人血小板中的特征。我们将确定L1分子是否存在于血小板中，以及它们是否具有逆转录酶活性。我们还确定L1元件是否控制着以前未知的血小板功能，包括将L1序列插入到线粒体DNA中，以及将mRNA逆转录到cDNA中。在AIM 2上的研究将检查新分离的血小板和储存的血小板的反应，否则将用于血小板输注。总之，这些研究将为人类巨核细胞和血小板的细胞活动提供新的见解。AIM 2的研究还将提供重要的临床信息，特别是关于服用逆转录酶抑制剂的艾滋病毒感染患者和接受血小板输注的患者。

DESCRIPTION (provided by applicant): The nucleus controls gene expression patterns that characterize the function of individual cells. Responses that occur in the nucleus, such as transcription and splicing of pre-mRNA, are elaborate processes that utilize many factors. Because platelets circulate without a nucleus, they have been stereotyped as a cell that lacks complexity and is devoid of nuclear functions. However, our group has recently shown that platelets carry a functional spliceosome and in response to activating signals, platelets splice pre- mRNA into mature, translatable messages. This result was unexpected because pre- mRNA splicing was previously thought to reside only in the nucleus. Possession of a functional spliceosome demonstrates remarkable specialization and intricate information transfer from parental megakaryocytes to terminally differentiated platelets. It also raises the possibility that megakaryocytes endow platelets with other novel gene expression pathways during thrombopoiesis. Recent evidence indicates that the long interspersed nuclear element (LINE-1; also referred to as L1 elements) family of retrotransposons harbor reverse transcriptase activity that regulates cellular growth, differentiation and gene expression. This suggests that L1 elements may regulate megakaryopoiesis and proplatelet formation. If transferred to proplatelets, L1 elements may also control functional responses in mature, circulating platelets. Whether L1 elements control megakaryocyte and platelet function, however, has not been tested. Therefore, in this R21 application we will explore the possibility that megakaryocytes and platelets possess functional L1 elements. In specific aim 1 we will characterize the L1 machinery in megakaryocytes. We will determine if L1 encoded proteins are present in human stem cell-derived megakaryocytes and if they regulate megakaryocyte differentiation and proplatelet formation. In aim 2 we will characterize the L1 machinery in mature, human platelets. We will determine if L1 elements are present in platelets and if they possess reverse transcriptase activity. We also determine if L1 elements control previously-unrecognized functions of platelets that include insertion of L1 sequences into mitochondrial DNA and reverse transcription of mRNA into cDNA. Studies in aim 2 will examine responses in freshly-isolated platelets and in stored platelets that would otherwise be used for platelet transfusions. Together these studies will provide novel insight into the cellular activities of human megakaryocytes and platelets. Studies in aim 2 will also provide important clinical information, especially in regards to HIV-infected patients who are medicated with reverse transcriptase inhibitors and patients who receive platelet transfusions.