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Elucidating the Inhibitory Mechanisms of RNA Triple Helices in Nuclear Decay

阐明 RNA 三螺旋在核衰变中的抑制机制

基本信息

批准号：
9535374
负责人：
JESSICA Ann BROWN
金额：
$ 24.56万
依托单位：
UNIVERSITY OF NOTRE DAME
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9535374
关键词：
Binding Biological Biological Assay Biological Models Cells Complementary DNA Complex Crystallization Development Educational process of instructing Electrophoretic Mobility Shift Assay Elements Endocrine Enzymes Excision Exonuclease Face Genetic Transcription Human Herpesvirus 8 Infection Interruption K-Series Research Career Programs Kinetics Laboratories Length Lung Adenocarcinoma Lytic Phase MALAT1 gene Malignant Neoplasms Mammalian Cell Measures Mediating Molecular Mutation Analysis Neoplasm Metastasis Neoplasms Normal Cell Nuclear Nuclear Decay Nuclear RNA Nucleotides Pathway interactions Phase Physiologic pulse Poly A Poly(A) Tail Poly(A)-specific ribonuclease Process Proteins RNA RNA Binding RNA Decay RNA Degradation Race Research Research Project Grants Role Secure Series Structure Techniques Time Training Transcript Universities Untranslated RNA Viral Virus Work X-Ray Crystallography anti-cancer therapeutic base cancer cell design experimental study in vivo insight knock-down mRNA Decay novel novel therapeutics nuclease prevent public health relevance skills triple helix

项目摘要

DESCRIPTION (provided by applicant): Polyadenylated nuclear (PAN) RNA is the most abundant lytic-phase transcript produced by the Kaposi's sarcoma-associated herpesvirus. Its high nuclear accumulation localizes to an expression and nuclear retention element (ENE). The U-rich internal loop of the PAN ENE sequesters the 32-poly(A) tail of PAN RNA into a triple helix (denoted as ENE+A) that protects the transcript from a novel rapid deadenylation-dependent nuclear RNA decay pathway. Similar ENE structures are formed by the 32-end sequences of two cellular long noncoding RNAs (lncRNAs): metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia beta (MENß). These lncRNAs are highly abundant in cancer cells despite lacking canonical poly(A) tails. Instead, 32-genomically-encoded A-rich tracts are sequestered by the ENEs of MALAT1 and MENß RNAs into proposed blunt-ended, triplexes. Because 3'->5' decay is typically initiated by an exonuclease binding to single-stranded RNA, the blunt-ends of cellular ENEs+A are likely subjected to different decay processes than the 32-poly(A) tails of viral ENEs+A. Moreover, the putative triplexes of cellular ENEs+A contain UA-U triples interrupted by G and C nucleotides that are absent in all viral ENEs. I hypothesize that the different viral and cellular ENE+A structures are degraded via distinct decay mechanisms. The molecular basis for how viral and cellular ENE+A structures differentially block nuclear RNA decay has not been investigated. The enzymatic components of the rapid nuclear RNA decay pathway are unknown for PAN RNA and MALAT1 except that PAN RNA is likely deadenylated by poly(A)-specific ribonuclease (PARN). In Aim 1, a series of cell-based assays will characterize the MALAT1 ENE+A in nuclear RNA decay, including time-resolved 32-RACE and RNA-based knockdown approaches to identify decay machinery. In Aim 2, kinetic assays will establish the mechanistic basis for how viral and cellular ENEs+A inhibit decay enzymes during the binding and nucleotide excision steps. In Aim 3, X-ray crystallography will be used to solve the structures of decay enzymes in complex with the PAN or MALAT1 ENE+A. Solving these structures will provide the first insights into how enzymes recognize and interact with triplexes. Initial kinetic and structural studies will use PARN and PAN ENE+A as a model system. Similar analyses will be performed with the MALAT1 ENE+A and its decay machinery uncovered in Aim 1. These experiments will elucidate the molecular mechanisms of how triple-helical RNA stabilization elements impede nuclear RNA decay while providing significant insights into the understudied pathways of nuclear lncRNA decay, the biological roles of RNA triplexes, and the mechanisms that stabilize MALAT1, a cancer-promoting lncRNA. This Career Development Award will allow me to develop my professional skills (e.g. classroom teaching) and to acquire specific technical training (e.g. X-ray crystallography) in the laboratories of Drs. Joan Steitz and Thomas Steitz at Yale University. These activities will enable me to secure an academic professorship and to build a top-tier research group.

描述（由申请人提供）：聚腺苷化核（PAN） RNA是由卡波西氏肉瘤相关疱疹病毒产生的最丰富的裂解期转录物。它的高核积累定位于一个表达和核保留元件（ENE）。PAN ENE富含u的内部环将PAN RNA的32 poly(A)尾部分离成三螺旋结构（表示为ENE+A），保护转录本免受新型快速死烯化依赖的核RNA衰变途径的影响。类似的ENE结构是由两种细胞长链非编码rna （lncRNAs）的32端序列形成的：转移相关肺腺癌转录本1 （MALAT1）和多发性内分泌瘤β （MENß）。这些lncrna在癌细胞中非常丰富，尽管缺乏典型的多聚(A)尾部。相反，32个基因组编码的富含a的束被MALAT1和MENß rna的ene隔离成钝端三联体。由于3‘->5’衰变通常是由与单链RNA结合的核酸外切酶引发的，因此细胞ENEs+A的钝端可能受到与病毒ENEs+A的32 poly(A)尾部不同的衰变过程。此外，假定的细胞ENEs+A三联体含有UA-U三联体，这些三联体被所有病毒ENEs中不存在的G和C核苷酸打断。我假设不同的病毒和细胞ENE+A结构通过不同的衰变机制被降解。病毒和细胞ENE+A结构如何不同地阻断核RNA衰变的分子基础尚未被研究。除了PAN RNA可能被聚(A)特异性核糖核酸酶（PARN）去脂化外，对于PAN RNA和MALAT1来说，核RNA快速衰变途径的酶学成分是未知的。在Aim 1中，一系列基于细胞的检测将表征核RNA衰变中的MALAT1 ENE+ a，包括时间分辨32-RACE和基于RNA的敲低方法来识别衰变机制。在目标2中，动力学分析将建立病毒和细胞ENEs+A在结合和核苷酸切除步骤中如何抑制衰变酶的机制基础。在Aim 3中，x射线晶体学将用于求解与PAN或MALAT1 ENE+A配合物中的衰变酶的结构。解决这些结构将提供对酶如何识别和与三联体相互作用的第一个见解。最初的动力学和结构研究将使用PARN和PAN ENE+A作为模型系统。将对Aim 1中发现的MALAT1 ENE+A及其衰变机制进行类似的分析。这些实验将阐明三螺旋RNA稳定元件如何阻碍核RNA衰变的分子机制，同时为尚未被研究的核lncRNA衰变途径、RNA三联体的生物学作用以及稳定MALAT1（一种促进癌症的lncRNA）的机制提供重要见解。这个职业发展奖将使我能够发展我的专业技能（如课堂教学），并在博士的实验室获得具体的技术培训（如x射线晶体学）。耶鲁大学的Joan Steitz和Thomas Steitz。这些活动将使我获得一个学术教授的职位，并建立一个一流的研究小组。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models.

使用植物模型揭示甲氨蝶呤和培美曲塞对丝氨酸羟甲基转移酶作用的结构基础。

DOI：
10.1038/s41598-019-56043-4
发表时间：
2019
期刊：
Scientific reports
影响因子：
4.6
作者：
Ruszkowski,Milosz;Sekula,Bartosz;Ruszkowska,Agnieszka;Contestabile,Roberto;Nogues,Isabel;Angelaccio,Sebastiana;Szczepaniak,Andrzej;Dauter,Zbigniew
通讯作者：
Dauter,Zbigniew

Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula: A Structural Characterization.