Reverse Transcriptase Activity in Megakaryocytes and Platelets

描述（由申请人提供）：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，如前mrna的转录和剪接，是一个复杂的过程，利用了许多因素。因为血小板在没有细胞核的情况下循环，它们一直被认为是一种缺乏复杂性和缺乏核功能的细胞。然而，我们的研究小组最近发现，血小板携带一个功能性剪接体，并响应激活信号，将前mRNA剪接成成熟的、可翻译的信息。这一结果是出乎意料的，因为以前认为mRNA前剪接只存在于细胞核中。拥有一个功能性剪接体表明了显著的特化和复杂的信息传递从亲代巨核细胞到终末分化的血小板。这也提出了巨核细胞在血小板形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，逆转录转座子家族的长散布核元件（LINE-1，也称为L1元件）具有逆转录酶活性，可调节细胞生长、分化和基因表达。这表明L1元素可能调节巨核生成和血小板形成。如果转移到血小板前，L1元素也可能控制成熟循环血小板的功能反应。然而，L1元素是否控制巨核细胞和血小板功能尚未得到证实。因此，在本R21应用中，我们将探讨巨核细胞和血小板具有功能性L1元素的可能性。在具体目标1中，我们将描述巨核细胞中的L1机制。我们将确定L1编码蛋白是否存在于人类干细胞衍生的巨核细胞中，以及它们是否调节巨核细胞分化和血小板形成。在目标2中，我们将描述成熟人类血小板中的L1机制。我们将确定血小板中是否存在L1元素以及它们是否具有逆转录酶活性。我们还确定了L1元件是否控制先前未被识别的血小板功能，包括L1序列插入线粒体DNA和mRNA逆转录成cDNA。目的2的研究将检查新鲜分离的血小板和储存的血小板的反应，否则这些血小板将用于血小板输注。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。目标2中的研究也将提供重要的临床信息，特别是关于使用逆转录酶抑制剂治疗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.