Trans-splicing in C. Elegans

描述(申请人提供)：操纵子是线虫基因组的一个重要特征。它们的转录本是经过3‘末端形成和反式剪接处理的多顺反子前mRNAs。这两个事件发生在操纵子基因之间非常接近的地方。虽然3‘末端的形成通常伴随着转录终止，但在操纵子中不是。我们已经确定了关键序列和许多负责执行这些事件的关键反式作用蛋白和SnRNAs，我们正在确定它们扮演什么角色以及如何发挥作用。反式剪接和防止转录终止所需的关键序列是位于3‘端切割位点下游约50bp的Ur元件。我们将确定在那里绑定了什么。我们最近成功地从操纵子中获得了几个模板，这些模板在线虫胚胎提取物中被正确地体外反式剪接。这表明正确的反式剪接是与RNA结合的蛋白质的一种特性，而不需要共转录的RNA处理。我们将利用体外系统来测试广泛的突变底物，以鉴定正确反式剪接所需的所有序列。然后，我们将确定在这些序列中起作用的大分子，并确定它们所扮演的角色。正常情况下，转录终止伴随着3‘末端的形成，但在操纵子中不是这样。我们将使用染色质IP来分析RNA聚合酶CTD在穿越操纵子时发生的磷酸化事件。这些研究应该为3‘端切割如何发生而不伴随操纵子的转录终止提供重要的新见解。这项工作还可以为转录终止的鱼雷模型提供强有力的支持或驳斥。我们还在研究反式剪接SnRNPs的蛋白质组分的作用。提出了涉及这些组分的突变体以及它们如何与功能相关基因中的突变体相互作用的功能研究。我们发现其中两种蛋白与一种新的SnRNA结合，Sm Y是目前唯一一种功能未知的SnRNA。我们将研究该RNA在反式剪接和操纵子前mRNA处理中的可能作用。我们假设这些蛋白质是从反式剪接后的分支中间产物中回收Sm蛋白所必需的，我们将使用体内和体外实验来测试这一模型。最后，我们设计了适当反式剪接所需基因突变的遗传筛查。公共卫生相关性这个项目的重点是线虫基因表达的基本方式，线虫是造成人类巨大疾病负担的生物，以及家畜和栽培植物。这里研究的过程，反式剪接，发生在线虫身上，而不是它们的宿主。人们希望，如果我们能够详细了解这一过程，它可能会提出干扰它的方法，这样线虫就会对宿主不敏感的处理方法敏感。此外，我们从这个项目中获得的基础知识对于了解基因表达的一般机制，特别是mRNAs的加工可能是重要的，这些基础知识可能为治疗导致许多遗传疾病的mRNA剪接缺陷的方法提供线索。

DESCRIPTION (provided by applicant): Operons are an important feature of the C. elegans genome. Their transcripts are polycistronic pre-mRNAs that are processed by 3' end formation and trans-splicing. These two events occur in close proximity between operon genes. Although 3' end formation is generally accompanied by transcription termination, in operons it is not. We have identified the key sequences and many of the key trans-acting proteins and snRNAs responsible for carrying out these events, and we are determining what roles they play and how. The key sequence required for both the trans-splicing and for preventing transcription termination is the Ur element that occurs ~50 bp downstream of the 3' end cleavage site. We will determine what binds there. We have recently succeeded in obtaining several templates from operons that are correctly trans-spliced in vitro in a C. elegans embryo extract. This indicates that correct trans-splicing is a property of proteins that bind to the RNA, rather than requiring co-transcriptional RNA processing. We will exploit the in vitro system to test a wide range of mutant substrates to identify all sequences required for correct trans-splicing. We will then identify the macromolecules that act at these sequences and determine what roles they play. Normally transcription termination accompanies 3' end formation, but in operons this cannot be the case. We will use chromatin IP to analyze what phosphorylation events occur to the RNA polymerase CTD as it traverses an operon. These studies should provide important new insight into how 3' end cleavage can occur without accompanying transcription termination in operons. This work could also provide strong support for, or refutation of, the torpedo model for transcription termination. We are also studying the roles of the protein components of the trans-splicing snRNPs. Functional studies involving mutants of these components and how they interact with mutants in functionally related genes are proposed. We have discovered that two of these proteins are bound to a novel snRNA, Sm Y, currently the only snRNA whose function is not known. We will study the possible role of this RNA in trans-splicing and operon pre-mRNA processing. We have hypothesized that these proteins are required for recycling the Sm proteins from the branched intermediates following trans-splicing, and we will test this model using both in vivo and in vitro experiments. Finally, we have devised a genetic screen for mutants in genes required for proper trans-splicing. PUBLIC HEALTH RELEVANCE This project focuses on basic means of gene expression in nematodes, organisms that are responsible for a huge disease burden in humans, as well as domestic animals and cultivated plants. The process under study here, trans-splicing, occurs in nematodes but not their hosts. It is hoped that if we can come to understand this process in detail it may suggest ways to interfere with it, such that the nematode would be sensitive to a treatment that the host is not. Besides this, the basic knowledge we obtain from this project may be important in understanding mechanisms of gene expression in general, especially processing of mRNAs, and this basic knowledge may provide clues for means to treat mRNA splicing defects responsible for many genetic diseases.