Natural Antisense and RNP-4F Expression in Drosophila

果蝇中的天然反义和 RNP-4F 表达

• 批准号: 6752654
• 负责人: JACK C VAUGHN
• 金额: $ 20.09万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752654
• 关键词: Drosophilidae; RNA binding protein; RNA interference; RNA splicing; antisense nucleic acid; developmental genetics; genetic transcription; invertebrate embryology; messenger RNA; precursor mRNA; protein isoforms; protein localization; ribonucleoproteins; yeast two hybrid system

DESCRIPTION (provided by applicant): Drosophila RNP4F is an ortholog to human p110, which functions with the pre-mRNA processing (PRP) protein class as a recycling factor to carry U4 and U6-snRNPs to the assembling spliceosome. Both inherited and acquired defects in premRNA processing are being increasingly recognized as causes of human diseases. A notable example of a relatively common (and also relevant to this proposal) human genetic disease which has been shown to be due to mutations in three different PRP genes is retinitis pigmentosa (RP), affecting 1 in 4000 individuals, being characterized by progressive retinal degeneration and eventually total blindness. Our previous work has shown that rnp-4f mRNAs undergo alternative splicing; which suggests that RNP-4F may have other functions, perhaps related to those carded out by the fully spliced gene product. We outline plans to characterize alternative RNP-4F proteins and their functions, and anticipate that the results may lead to discovery of corresponding alternative p110 functions in humans. Specifically, we propose to: (a) fully identify all rnp.4f transcript isoforms and determine their in situ localizations; (b) determine in situ localizations for the correspondingly encoded proteins; (c) eliminate each mRNA isoform specifically using RNAi in an attempt to determine functions for the encoded RNP-4F isoforms; and (d) identify neighbor proteins for the different RNP-4F isoforms via yeast two-hybrid screens. We have previously reported existence of extensively A-to-G edited rnp-4f mRNA in an adult head cDNA clone, which was subsequently shown to have arisen due to the action of dADAR editase on double-stranded RNA in which the antisense strand was supplied by a developmentally regulated readthrough transcript class of the sas10 gene. A decline in rnp-4f mRNA levels was observed to accompany rising readthrough transcript levels, suggesting a role for the antisense RNA in posttranscriptional regulation of rnp-4f gene expression during development. In this proposal, we outline plans to determine the relationship between long natural antisense transcripts and gene expression control in the Drosophila rnp-4f/sas10 model gene pair. We will do this by: (a) showing that sas10 readthrough transcripts are required for the observed decline in rnp-4f mRNA levels; (b) showing that sas10 readthrough transcripts co-localize with rnp-4f mRNAs; and (c) evaluating mechanisms for rnp-4f decay: (1) dADAR editase transcript modification followed by degradation and (2) destruction via the RNAi pathway. This research will provide a basis for understanding the role of long natural antisense RNAs in posttranscriptional control of eukaryotic gene expression, and may provide a model for elucidation of roles for comparable RNAs in organisms including humans, estimated to contain about 800 such transcripts. This work will also lead: to elucidation of alternative RNP-4F functions: perhaps to discovery of corresponding alternative p110 functions in humans.

描述（由申请人提供）：果蝇RNP4F是人类p110的直系同源物，它与MRNA加工前（PRP）蛋白类别一起起作用，是将U4和U6-SNRNP携带到组装剪接体的回收因子。在premrna处理中，遗传和获得的缺陷越来越被认为是人类疾病的原因。一个相对常见的人类遗传疾病的一个值得注意的例子（也与该提案相关），该疾病已证明是由于三种不同的PRP基因突变引起的，是色素性视网膜炎（RP），影响4000个个体中的1个，其特征是进行性视网膜退化和最终的全盲。我们以前的工作表明，RNP-4F mRNA经历了替代剪接。这表明RNP-4F可能具有其他功能，也许与完全剪接的基因产物夹住的功能有关。 我们概述了表征替代RNP-4F蛋白及其功能的计划，并预测结果可能会导致人类中相应的替代p110功能。具体而言，我们建议：（a）充分识别所有RNP.4F转录本同工型并确定其原位定位； （b）确定相应编码的蛋白质的原位定位； （c）消除每个mRNA同工型，特异性使用RNAi，以确定编码的RNP-4F同工型的功能； （d）通过酵母两杂交筛网鉴定不同RNP-4F同工型的邻居蛋白。我们先前曾报道过在成年头部cDNA克隆中存在广泛的A到G编辑的RNP-4F mRNA，随后由于达达尔编辑酶对双链链RNA的作用而显示出出现，在该RNA上，反义链由SAS10 Gene的开发调节的读取型转录类提供。观察到RNP-4F mRNA水平的下降伴随着读取的转录水平上升，这表明反义RNA在发育过程中RNP-4F基因表达的转录后调节中起作用。在此提案中，我们概述了果蝇RNP-4F/SAS10模型基因对中长的自然反义转录本与基因表达控制之间的关系。我们将通过：（a）表明SAS10读取的转录本是RNP-4F mRNA水平下降所必需的； （b）表明SAS10读取转录本与RNP-4F mRNA共定位； （c）评估RNP-4F衰减的机制：（1）DADAR编辑酶的转录本修改，然后降解和（2）通过RNAi途径破坏。这项研究将为理解长自然反义RNA在真核基因表达的转录后控制中的作用提供基础，并可能为阐明包括人类在内的生物体中可比RNA的作用提供模型，包括人类，估计包含约800个此类转录本。这项工作还将导致：阐明替代性RNP-4F功能：可能发现人类中相应的替代p110功能。