Novel tools to investigate local and global RNA conformations in the spliceosome
研究剪接体中局部和整体 RNA 构象的新工具
基本信息
- 批准号:9164146
- 负责人:
- 金额:$ 9万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2016
- 资助国家:美国
- 起止时间:2016-09-16 至 2018-08-31
- 项目状态:已结题
- 来源:
- 关键词:5&apos Splice SiteAddressAdenosineAdoptedAdvisory CommitteesAlternative SplicingAwardBindingBiochemicalBiologicalBiophysicsCircular DichroismCircular Dichroism SpectroscopyCodeComplexCoupledCouplingDNADefectDetectionDiseaseElectronicsEnergy TransferEukaryotaEventExcisionExcitonExhibitsExonsFluorescenceFluorescence Resonance Energy TransferGene ExpressionGenesGenetic TranscriptionGoalsHereditary DiseaseHumanHuman GeneticsInstitutionIntronsJoining ExonsLabelLaboratoriesLengthLightMacromolecular ComplexesMeasurementMeasuresMentorsMessenger RNAMethodsMichiganMicroscopyModelingMolecularMolecular BiologyMolecular ConformationMutationNucleic Acid ConformationNucleotidesOpen Reading FramesPathway interactionsPeptide Nucleic AcidsPhaseProcessProtein BiosynthesisProteinsProtocols documentationRNARNA ConformationRNA HelicaseRNA SplicingRegulationReportingResearchRoleRunningSamplingScientistSignal TransductionSiteSmall Nuclear RNASmall Nuclear RibonucleoproteinsSpectrum AnalysisSpliceosomesStructureSystemT7 RNA polymeraseTechniquesTestingTrainingU5 small nuclear RNAUniversitiesWorkanalogbasebiological systemscareercareer developmentchromophorecyanine dye 5dimerfluorophorehuman diseaseinstrumentationinteinliterature surveymRNA Precursornovelpost-doctoral trainingpreventresearch studyresponsesingle moleculetherapy developmenttool
项目摘要
Project Summary
In eukaryotes, the vast majority of genes have their protein-coding regions (exons) split up, separated by
introns containing up to tens of thousands of nucleotides. The removal of introns, called “splicing”, is a critical
step in gene expression that allows for exquisitely fine-tuned regulation and, through alternative splicing,
diversifies a single gene into more than one protein. Splicing is executed by the spliceosome, a multi-
megaDalton macromolecular complex whose function requires interactions between the pre-messenger RNA
(pre-mRNA) substrate, five small nuclear ribonucleoprotein particles (snRNPs), and numerous additional
protein factors. Determining the roles of and interactions between these components is of central importance to
understanding the molecular mechanisms of the many human diseases in which aberrant splicing is
implicated.
The recent application of single-molecule microscopy to the spliceosome has shed much light on the
molecular mechanism of splicing. However, the interactions between the snRNAs and the pre-mRNA have
remained difficult to probe due to the challenge of preparing snRNAs that are site-specifically fluorophore-
labeled. Furthermore, conformational changes can be tracked only on certain length scales, limited by the
sensitivity of the experimental techniques used, which are often based on Förster resonance energy transfer
(FRET). To address these challenges, Specific Aim 1 will study the rearrangement of interactions between U5
snRNA and the pre-mRNA in response to the action of RNA helicase Prp22. Site-specifically fluorophore-
labeled U5 will be prepared through by using a short peptide nucleic acid oligomer to stall transcription by T7
RNA polymerase at the desired labeling site, a general approach that avoids many of the downsides of other
RNA labeling methods. Specific Aim 2 proposes the novel technique of FRET-filtered spectroscopy (FFS),
which will utilize two closely spaced fluorophores as a FRET donor, and an additional fluorophore as an
acceptor. FFS will use electronic coupling between the two donors to reveal their local conformation as a
function of their distance from the acceptor, and can be expanded to utilize any type of fluorescence-detected
spectroscopy as a readout. This technique will be applied to Cy3- and Cy5-labeled RNA to study the unwinding
of RNA duplexes by Prp22. Specific Aim 3 combines the labeling method of Aim 1 with FFS, utilizing FRET-
filtered circular dichroism spectroscopy to determine the changes in local pre-mRNA conformation in the
vicinity of the branchpoint adenosine as purified Bact intermediates are chased through the first step of splicing.
This work will answer longstanding questions about the correlations between local and global RNA
conformations in the spliceosome, and involves novel methods that can be generalized to many different
biological systems. Aim 1 and the initial experiments for Aim 2 will be pursued in the laboratory of the
applicant's research mentor, while Aim 2 will be completed and Aim 3 will be both initiated and completed in
the applicant's independent laboratory.
During the mentored phase of the award, the applicant will be working at the University of Michigan in
the laboratory of Dr. Nils Walter, who has a strong record of training successful scientists. The applicant has
assembled an advisory committee who, together with Dr. Walter, will provide guidance on her research and her
transition into an independent career. The applicant's career goals involve running an independent laboratory
at an academic institution, and she seeks to combine her graduate training in spectroscopy with her ongoing
postdoctoral training in RNA molecular biology and biophysics. In addition to providing the instrumentation
necessary for the proposed research, the University of Michigan hosts numerous organizations and events that
will contribute to the applicant's training and career development. This proposal builds on all of the applicant's
previous and ongoing training to open a unique window into the function of the spliceosome.
项目摘要
在真核生物中,绝大多数基因的蛋白质编码区(外显子)被分开,
内含子含有多达数万个核苷酸。内含子的去除,称为“剪接”,是一个关键的
基因表达中的一个步骤,允许精确的微调调节,通过选择性剪接,
将一个基因分化成一种以上的蛋白质。剪接是由剪接体,一个多-
一种百万道尔顿的大分子复合物,其功能需要前信使RNA之间的相互作用
(前mRNA)底物,五个小的核核糖核蛋白颗粒(snRNP),和许多额外的
蛋白质因素确定这些组成部分的作用和相互作用对以下方面至关重要:
了解许多人类疾病的分子机制,其中异常剪接是
牵连
最近单分子显微镜在剪接体上的应用,使人们对剪接体的结构有了更多的了解。
剪接的分子机制。然而,snRNA和前mRNA之间的相互作用,
由于制备位点特异性荧光团的snRNA的挑战,
标签。此外,构象变化只能在一定的长度尺度上被追踪,这受限于分子量。
使用的实验技术的灵敏度,这往往是基于福斯特共振能量转移
(FRET).为了应对这些挑战,具体目标1将研究U 5之间相互作用的重新排列
snRNA和前mRNA响应RNA解旋酶Prp 22的作用。位点特异性荧光团-
通过使用短肽核酸寡聚体来阻止T7的转录,将制备标记的U 5
RNA聚合酶在所需的标记位点,一个通用的方法,避免了许多其他的缺点,
RNA标记方法。具体目标2提出了FRET滤波光谱(FFS)的新技术,
其将利用两个紧密间隔的荧光团作为FRET供体,并且另外的荧光团作为FRET供体。
接受者FFS将利用两个供体之间的电子耦合来揭示它们作为一种分子的局部构象。
它们与受体的距离的函数,并且可以扩展到利用任何类型的荧光检测
光谱作为读数。该技术将应用于Cy 3和Cy 5标记的RNA以研究解旋
Prp 22的RNA双链体。特异性目标3将目标1的标记方法与FFS结合,利用FRET-
过滤的圆二色光谱,以确定在局部前mRNA构象的变化,
作为纯化的Bact中间体的分支点附近的腺苷被追踪通过剪接的第一步。
这项工作将回答长期存在的关于局部和全局RNA之间相关性的问题
剪接体中的构象,并涉及新的方法,可以推广到许多不同的
生物系统。目标1和目标2的初步实验将在实验室进行,
申请人的研究导师,而目标2将完成,目标3将启动和完成,
申请人的独立实验室。
在该奖项的指导阶段,申请人将在密歇根大学工作,
尼尔斯·沃尔特博士的实验室,他在培养成功的科学家方面有着良好的记录。申请人已经
组建了一个咨询委员会,该委员会将与沃尔特博士一起为她的研究和她的研究提供指导。
过渡到独立的职业生涯。申请人的职业目标包括经营一个独立的实验室
在一个学术机构,她寻求联合收割机结合她的光谱学研究生培训,她正在进行的
RNA分子生物学和生物物理学博士后培训。除了提供仪器外,
为了进行拟议的研究,密歇根大学举办了许多组织和活动,
将有助于申请人的培训和职业发展。该提案建立在申请人的所有
以前和正在进行的训练,以打开一个独特的窗口进入剪接体的功能。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Julia Reed Widom其他文献
Julia Reed Widom的其他文献
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{{ truncateString('Julia Reed Widom', 18)}}的其他基金
Mapping the sequence landscape of RNA structure, dynamics and protein interactions using high-throughput single-molecule FRET
使用高通量单分子 FRET 绘制 RNA 结构、动力学和蛋白质相互作用的序列图谱
- 批准号:
10707257 - 财政年份:2022
- 资助金额:
$ 9万 - 项目类别:
Novel Tools to Investigate Local and Global RNA Conformations in the Spliceosome
研究剪接体中局部和整体 RNA 构象的新工具
- 批准号:
9814290 - 财政年份:2016
- 资助金额:
$ 9万 - 项目类别:
Novel Tools to Investigate Local and Global RNA Conformations in the Spliceosome
研究剪接体中局部和整体 RNA 构象的新工具
- 批准号:
10093064 - 财政年份:2016
- 资助金额:
$ 9万 - 项目类别:
Novel tools to investigate local and global RNA conformations in the spliceosome
研究剪接体中局部和整体 RNA 构象的新工具
- 批准号:
9353434 - 财政年份:2016
- 资助金额:
$ 9万 - 项目类别:
Dissecting the Functions of RNA Helicases in Single Spliceosomes
剖析单剪接体中 RNA 解旋酶的功能
- 批准号:
8830784 - 财政年份:2015
- 资助金额:
$ 9万 - 项目类别:
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