Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins
PTB 和 Rbfox 蛋白转录后基因调控机制
基本信息
- 批准号:10589873
- 负责人:
- 金额:$ 77.81万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-04-01 至 2025-03-31
- 项目状态:未结题
- 来源:
- 关键词:Alternative SplicingAmyotrophic Lateral SclerosisArchitectureBindingBinding SitesBiochemicalBiochemistryBiologicalBiological ProcessBiologyCell LineCellsChromatinClustered Regularly Interspaced Short Palindromic RepeatsComplexCytoplasmDevelopmentDiseaseEpilepsyEventExonsFamilyFrontotemporal DementiaFunctional disorderGene ExpressionGenesIntronsInvestigationLasR proteinMapsMass Spectrum AnalysisMediatingMethodsMolecularMolecular Mechanisms of ActionMyotonic DystrophyNervous SystemNeurodegenerative DisordersNeuronsNuclearPhysical condensationPolypyrimidine Tract-Binding ProteinPost-Transcriptional RegulationProductivityProtein FamilyProtein IsoformsProteinsRNARNA SplicingRNA-Binding ProteinsReactionRegulationRepressionRoleSpinal Muscular AtrophySpliceosome Assembly PathwaySubcellular FractionsSynapsesTranslationsWorkautism spectrum disorderdifferentiation protocolfollow-upgenome editinggenome-widehuman diseasemRNA PrecursormRNA Stabilitynervous system disorderneuron developmentposttranscriptionalprogramsstem cell differentiationtargeted treatmenttranscriptometranscriptome sequencing
项目摘要
PROJECT SUMMARY/ABSTRACT
This MIRA application is to support studies of how RNA binding proteins regulate choices in alternative splicing
and other posttranscriptional steps in mammalian gene expression. We will continue our studies of two families
of regulators, the Polypyrimidine Tract Binding Proteins and the Rbfox proteins. We will examine their
molecular mechanisms of action, their biological functions, and the roles of their extended regulatory programs
in neuronal development and mature neuronal function. Multiple human diseases, including several
neurodegenerative disorders, involve the dysfunction of RNA binding proteins and aberrant splicing regulation.
To develop treatments for such disorders, we need greater understanding of both the mechanisms and the
biology of alternative splicing. We will continue our studies of the nuclear and cytoplasmic Rbfox isoforms. We
will apply biochemistry and genome edited cell lines to examine how the eight RNA binding proteins of the
LASR complex bind with each other, and how the assembled complex interacts with nuclear Rbfox to regulate
splicing. RNAseq analyses of purified complexes and genomewide iCLIP analyses will map the binding of
LASR subunits relative to the known Rbfox binding sites to reveal how the RNA within the LASR complex is
organized. We will follow up on recent studies of cytoplasmic Rbfox isoforms to examine how these proteins
regulate the translation and stability of mRNAs encoding important synaptic proteins, such as Vamp1. We will
also continue our analyses of the Rbfox intrinsically disordered region and its ability to form molecular
condensates. These analyses will be extended to IDR's in the LASR subunits to examine their homotypic and
heterotypic interactions, and the role of their condensation in splicing regulation. Our studies of the
mechanisms and biology of splicing repression by PTBP1 and PTBP2 will be continued. We will use
biochemical methods developed in earlier work and new mass spectrometry approaches to examine the
assembly and architecture of exon complexes repressed by PTBP1 and understand how PTBP1 blocks
productive spliceosome assembly. We will extend our investigation of the biological impact of two transitions in
neuronal splicing regulation: one induced early in neuronal development when PTBP1 is replaced with PTBP2,
and one occurring when PTBP2 is downregulated late in neuronal maturation. The roles of particular splicing
switches within the PTBP programs will be examined using stem cell differentiation protocols, CRISPR
mediated gene editing, and whole transcriptome expression and splicing analyses. Applying RNAseq to
subcellular fractions, we will characterize intron retention events controlled by PTBP1 and examine the
mechanisms that sequester RNAs on chromatin. Altogether these studies will yield new understanding of the
intricate molecular interactions that mediate the regulation of splicing and its misregulation in human disease.
项目总结/摘要
这个MIRA应用程序是为了支持RNA结合蛋白如何调节选择性剪接的研究
以及哺乳动物基因表达中的其他转录后步骤。我们将继续研究两个家庭
调节剂,多嘧啶道结合蛋白和Rbfox蛋白。我们将检查他们的
分子作用机制,它们的生物学功能,以及它们的扩展调节程序的作用
在神经元发育和成熟神经元功能中。多种人类疾病,包括
神经退行性疾病涉及RNA结合蛋白的功能障碍和异常剪接调节。
为了开发治疗这些疾病的方法,我们需要更好地了解这些疾病的机制和治疗方法。
选择性剪接的生物学我们将继续我们的核和细胞质Rbfox亚型的研究。我们
将应用生物化学和基因组编辑的细胞系来研究细胞中的八种RNA结合蛋白是如何与基因组结合的。
LASR复合物相互结合,以及组装的复合物如何与核Rbfox相互作用以调节
拼接纯化的复合物的RNAseq分析和全基因组iCLIP分析将绘制结合蛋白的图谱。
LASR亚基相对于已知的Rbfox结合位点的结合,以揭示LASR复合物内的RNA是如何被结合的。
有组织的。我们将跟进最近的研究细胞质Rbfox亚型,以检查这些蛋白质如何
调节编码重要突触蛋白(如Vamp 1)的mRNA的翻译和稳定性。我们将
我们还继续分析了Rbfox的本征无序区及其形成分子的能力
冷凝物这些分析将扩展到LASR亚基中的IDR,以检查它们的同型性,
异型相互作用,以及它们的缩合在剪接调节中的作用。我们的研究
PTBP 1和PTBP 2的剪接抑制机制和生物学将继续进行。我们将使用
在早期工作中开发的生物化学方法和新的质谱方法来检查
PTBP 1抑制的外显子复合物的组装和结构,并了解PTBP 1如何阻断
生产性剪接体组装。我们将扩大我们的调查的生物影响的两个转变,
神经元剪接调节:当PTBP 1被PTBP 2取代时,
另一种发生在PTBP 2在神经元成熟后期下调时。特定剪接的作用
PTBP项目中的开关将使用干细胞分化方案,CRISPR,
介导的基因编辑以及全转录组表达和剪接分析。应用RNAseq
亚细胞组分,我们将表征由PTBP 1控制的内含子保留事件,并检查
在染色质上隔离RNA的机制。总之,这些研究将产生新的认识,
在人类疾病中介导剪接调节及其失调的复杂分子相互作用。
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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{{ truncateString('Douglas L Black', 18)}}的其他基金
Comprehensive Maps of U1 snRNP Binding to Nascent RNA in Human Cells
U1 snRNP 与人类细胞中新生 RNA 结合的综合图谱
- 批准号:
10507429 - 财政年份:2022
- 资助金额:
$ 77.81万 - 项目类别:
Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins
PTB 和 Rbfox 蛋白转录后基因调控机制
- 批准号:
10362546 - 财政年份:2020
- 资助金额:
$ 77.81万 - 项目类别:
Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins
PTB 和 Rbfox 蛋白转录后基因调控机制
- 批准号:
10797969 - 财政年份:2020
- 资助金额:
$ 77.81万 - 项目类别:
Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins
PTB 和 Rbfox 蛋白转录后基因调控机制
- 批准号:
10810036 - 财政年份:2020
- 资助金额:
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Multi-omic analysis of Myc-driven splicing for prostate cancer therapeutic development
Myc 驱动剪接的多组学分析用于前列腺癌治疗开发
- 批准号:
9898152 - 财政年份:2018
- 资助金额:
$ 77.81万 - 项目类别:
Multi-omic analysis of Myc-driven splicing for prostate cancer therapeutic development
Myc 驱动剪接的多组学分析用于前列腺癌治疗开发
- 批准号:
10364684 - 财政年份:2018
- 资助金额:
$ 77.81万 - 项目类别:
Elucidating an Xist-dependent program of sexually dimorphic alternative splicing in the mammalian brain
阐明哺乳动物大脑中依赖于 Xist 的性二态选择性剪接程序
- 批准号:
9305157 - 财政年份:2016
- 资助金额:
$ 77.81万 - 项目类别:
Elucidating an Xist-dependent program of sexually dimorphic alternative splicing in the mammalian brain
阐明哺乳动物大脑中依赖于 Xist 的性二态选择性剪接程序
- 批准号:
9922380 - 财政年份:2016
- 资助金额:
$ 77.81万 - 项目类别:
Mechanisms of Alternative Splicing Regulation by Rbfox Proteins
Rbfox 蛋白的选择性剪接调控机制
- 批准号:
9353837 - 财政年份:2016
- 资助金额:
$ 77.81万 - 项目类别:
Mechanisms of Alternative Splicing Regulation by Rbfox Proteins
Rbfox 蛋白的选择性剪接调控机制
- 批准号:
9175889 - 财政年份:2016
- 资助金额:
$ 77.81万 - 项目类别:
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