Trans-splicing in C. Elegans

线虫中的反式剪接

• 批准号: 8135028
• 负责人: Thomas Blumenthal
• 金额: $ 35.53万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8135028
• 关键词: Affect; Affinity; Ascaris; Base Pairing; Binding; Bioinformatics; C. elegans genome; Caenorhabditis elegans; Chromatin; Consensus; DNA Polymerase II; DNA-Directed RNA Polymerase; Defect; Domestic Animals; Elements; Embryo; Enzymes; Event; Exonuclease; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Health; Hereditary Disease; Human; In Vitro; Knowledge; Messenger RNA; Modeling; Nematoda; Operon; Organism; Pattern; Phosphorylation; Plants; Play; Post-Translational Protein Processing; Process; Property; Protein Binding; Proteins; RNA; RNA Interference; RNA Processing; RNA Splicing; Recycling; Role; Signal Transduction; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Specificity; Spliced Leader RNA; Spliced Leader Sequences; System; Testing; Time; Torpedo; Trans-Splicing; Transcript; Work; Yeasts; burden of illness; chromatin immunoprecipitation; in vivo; insight; mRNA Precursor; macromolecule; man; mutant; novel; paralogous gene; prevent; promoter; protein complex; research study; snRNP Structural Core Protein; transcription termination

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.

描述（由申请人提供）：操纵子是C。线虫基因组它们的转录物是通过3'末端形成和反式剪接加工的多顺反子前mRNA。这两个事件发生在操纵子基因之间非常接近。虽然3'末端的形成通常伴随着转录终止，但在操纵子中并非如此。我们已经确定了负责执行这些事件的关键序列和许多关键的反式作用蛋白质和snRNAs，我们正在确定它们发挥什么作用以及如何发挥作用。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50 bp处的Ur元件。我们将决定什么约束那里。我们最近成功地从操纵子中获得了几个模板，这些操纵子在体外C。线虫胚胎提取物。这表明正确的反式剪接是蛋白质与RNA结合的特性，而不需要共转录RNA加工。我们将利用体外系统测试广泛的突变底物，以确定正确的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，并确定它们扮演的角色。通常转录终止伴随3'末端形成，但在操纵子中不是这种情况。我们将使用染色质IP来分析RNA聚合酶CTD在穿过操纵子时发生的磷酸化事件。这些研究应该提供重要的新的见解如何3'端切割可以发生，而不伴随转录终止操纵子。这项工作也可以提供有力的支持，或反驳，鱼雷模型的转录终止。我们还在研究反式剪接snRNP的蛋白质组分的作用。功能研究涉及这些组件的突变体，以及它们如何与功能相关基因的突变体相互作用。我们发现其中两种蛋白质与一种新的snRNA Sm Y结合，Sm Y是目前唯一一种功能未知的snRNA。我们将研究这种RNA在反式剪接和操纵子前mRNA加工中的可能作用。我们假设，这些蛋白质是需要回收的Sm蛋白质从分支中间体后反式剪接，我们将测试这个模型使用在体内和体外实验。最后，我们设计了一个遗传筛选所需的适当的反式剪接的基因突变体。 公共卫生相关性本项目的重点是线虫基因表达的基本方法，线虫是人类以及家畜和栽培植物中造成巨大疾病负担的生物体。这里研究的过程，反式剪接，发生在线虫中，但不是它们的宿主。希望如果我们能够详细了解这个过程，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敏感。除此之外，我们从这个项目中获得的基本知识可能对理解基因表达的机制很重要，特别是mRNA的加工，这些基本知识可能为治疗导致许多遗传疾病的mRNA剪接缺陷提供线索。

项目成果

SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5' end of Caenorhabditis elegans pre-mRNA.

SL1 反式剪接由插入到秀丽隐杆线虫前体 mRNA 5 端的富含 AU 的合成 RNA 指定。

• 发表时间: 1995
• 期刊: RNA (New York, N.Y.)
• 作者: Conrad,R;Lea,K;Blumenthal,T
• 通讯作者: Blumenthal,T

U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans.

• DOI: 10.1017/s1355838299982225
• 发表时间: 1999-04
• 期刊: RNA
• 影响因子: 4.5
• 作者: D. Zorio;T. Blumenthal

TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA.

• DOI: 10.15252/embj.201488740
• 发表时间: 2014-12-17
• 期刊: The EMBO journal
• 作者: Saldi TK;Ash PE;Wilson G;Gonzales P;Garrido-Lecca A;Roberts CM;Dostal V;Gendron TF;Stein LD;Blumenthal T;Petrucelli L;Link CD
• 通讯作者: Link CD

Relationship between 3' end formation and SL2-specific trans-splicing in polycistronic Caenorhabditis elegans pre-mRNA processing.

• 发表时间: 1997-03
• 期刊: RNA
• 影响因子: 4.5
• 作者: S. Kuersten;K. Lea;M. MacMorris;J. Spieth;T. Blumenthal

Functional analysis of an intron 3' splice site in Caenorhabditis elegans.

秀丽隐杆线虫内含子 3 剪接位点的功能分析。

• 发表时间: 1996
• 期刊: RNA (New York, N.Y.)
• 作者: Zhang,H;Blumenthal,T
• 通讯作者: Blumenthal,T