Methods for purification of individual nuclear pre-messenger RNA-protein complexe

描述（由申请人提供）：本提案试图开发纯化和表征单个核前mrna -蛋白复合物的方法。由于真核生物RNA结合蛋白与新生转录物的结合发生在细胞核中，因此人们认为，特定转录物在形成独特的核糖核蛋白（RNP）复合体时获得的特定RNA结合蛋白群将控制其RNA加工、RNA输出和RNA稳定性。这些转录后通路对基因表达至关重要。此外，由于95%的人类基因产生多种转录异构体，因此发现不同剪接的mRNA异构体是否具有不同的RNA结合蛋白模式也很重要。正在测试的假设是，不同的核前mrna /RNPs具有不同的蛋白质组成，这决定了它们的命运。如果成功，这种方法将对我们理解核RNP结构如何控制RNA加工和新转录信使RNA分子的命运产生重大影响。为了解决这个问题，我们将：1。开发针对特定前mrna的生物素化反义嵌合na - dna寡核苷酸纯化单个果蝇核前mrna的技术，并通过质谱分析其蛋白质组成。本提案将概述一个特定的目标，该目标建立在我的实验室在评估备选前mrna剪接的全基因组模式和核前mrna上RNA剪接因子的分布方面的专业知识之上。

DESCRIPTION (provided by applicant): This proposal attempts to develop methods for the purification and characterization of individual nuclear pre-mRNA-protein complexes. Because binding of eukaryotic RNA binding proteins to nascent transcripts occurs in the nucleus during transcription, it is believed that the particular constellation of RNA binding proteins that a given transcript acquires to form a distinct ribonucleoprotein (RNP) complex will control its RNA processing, RNA export and RNA stability. These post-transcriptional pathways are critically important for gene expression. Moreover, because >95% of human genes generate multiple transcript isoforms it is also important to find out if differentially spliced mRNA isoforms have distinct patterns of RNA binding proteins. The hypothesis being tested is that different nuclear pre-mRNAs/RNPs have distinct protein compositions which contribute to their fate. If successful, this approach would have a major impact in our understanding of how nuclear RNP structure controls the RNA processing and fates of newly transcribed messenger RNA molecules. In order to approach this question, we will: 1. Develop technology to purify individual Drosophila nuclear pre-mRNPs using biotinylated anti-sense chimeric LNA-DNA oligonucleotides targeted to specific pre-mRNAs and analyze their protein composition by mass spectrometry. This proposal will outline one specific aim that builds on the expertise of my lab in evaluating genome-wide patterns of alternative pre-mRNA splicing and the distribution of RNA splicing factors on nuclear pre-mRNAs. PUBLIC HEALTH RELEVANCE: This proposal attempts to develop methods for the purification and characterization of individual nuclear pre-mRNA-protein complexes. Because binding of eukaryotic RNA binding proteins to nascent transcripts occurs in the nucleus during transcription, it is believed that the particular constellation of RNA binding proteins that a given transcript acquires to form a distinct ribonucleoprotein (RNP) complex will control its RNA processing, RNA export and RNA stability. These post-transcriptional pathways are critically important for gene expression and can be perturbed in disease states.