Trans-splicing in C. Elegans

线虫中的反式剪接

基本信息

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
项目类别：
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

项目摘要

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）：操纵子是秀丽隐杆线虫基因组的重要特征。它们的转录本是通过3'末端形成和移媒体进行处理的多用电话preciston。这两个事件发生在操纵子基因之间的近端。尽管3'末端的形成通常伴随着转录终止，但在操纵子中却没有。我们已经确定了关键序列以及负责执行这些事件的许多关键跨作用蛋白和SNRNA，我们正在确定它们扮演的角色以及如何扮演。跨切割和防止转录终止所需的关键序列是3'端裂解位点下游约50 bp的UR元素。我们将确定那里的约束。最近，我们成功地从秀丽隐杆线虫胚胎提取物中在体外正确地进行了切割的操纵子中获取了几个模板。这表明正确的移序是与RNA结合的蛋白质的特性，而不是需要共转录RNA处理。我们将利用体外系统来测试各种突变底物，以识别正确的移植所需的所有序列。然后，我们将确定以这些序列作用的大分子，并确定它们扮演的角色。通常，转录终止伴随3'结束构成，但在操纵子中并非如此。我们将使用染色质IP来分析RNA聚合酶CTD发生的磷酸化事件，因为它遍历了操纵子。这些研究应该为如何在不伴随操纵子中的转录终止而进行3'端裂解的重要新见解。这项工作还可以为转录终止的鱼雷模型提供大力支持或反驳。我们还研究了跨式SNRNP的蛋白质成分的作用。提出了涉及这些成分突变体以及它们如何与功能相关基因中突变体相互作用的功能研究。我们已经发现，这些蛋白质中的两种与新的SnRNA结合，目前是唯一尚不清楚功能的snRNA。我们将研究该RNA在变速器和操纵子前MRNA处理中的可能作用。我们假设这些蛋白质是从分类后从分支中间体中回收SM蛋白所必需的，我们将使用体内和体外实验测试该模型。最后，我们设计了一个基因中的突变体的基因筛选，以适当的移植。公共卫生相关性该项目着重于线虫中基因表达的基本手段，导致人类造成巨大疾病负担的生物以及家畜和栽培植物。这里研究的过程是在线虫中发生的，而不是其宿主发生的。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，以便线虫对宿主不这样做的治疗敏感。除此之外，我们从该项目中获得的基本知识可能在理解一般的基因表达机制，尤其是mRNA的处理机制中很重要，并且这种基本知识可能为治疗负责许多遗传疾病的mRNA剪接缺陷提供了方法。

项目成果

期刊论文数量（25）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
通讯作者：
D. Zorio;T. Blumenthal

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。

DOI：
发表时间：
1995
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Conrad,R;Lea,K;Blumenthal,T
通讯作者：
Blumenthal,T

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
影响因子：
0
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.