Addressing Protein Synthesis Regulation within Small Numbers of Discrete Neurons
解决少量离散神经元内的蛋白质合成调控问题
基本信息
- 批准号:10586226
- 负责人:
- 金额:$ 40.63万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2013
- 资助国家:美国
- 起止时间:2013-09-30 至 2028-01-31
- 项目状态:未结题
- 来源:
- 关键词:AddressAdultAffinity ChromatographyAnimalsAreaBiochemicalBiochemistryBiocompatible MaterialsBiologicalBiological AssayBiologyBrainBrain DiseasesCatalytic DomainCell Culture SystemCell Culture TechniquesCell LineCellsChimeric ProteinsCircadian RhythmsCircadian gene expressionCommunitiesComplementComputing MethodologiesDataDevelopmentDiseaseDrosophila genusEnzymesFractionationFragile X SyndromeFutureGene ExpressionGene Expression RegulationGeneticGenetic TranscriptionHumanIn VitroKnock-outLabor ComplicationsLaboratory StudyLateralMalariaMalignant NeoplasmsMammalian CellMammalsMedicineMessenger RNAMethodsMusNeurodegenerative DisordersNeuronsNeurosciencesNoiseOrganismPacemakersParasitesPersonsPharmaceutical PreparationsPlantsPlasmidsPolyribosomesPopulationPost-Transcriptional RegulationProtein BiosynthesisProteinsRNARNA EditingRNA-Binding ProteinsRecombinant ProteinsRecombinantsRegulationResearch PersonnelRibosomal ProteinsRibosomesRoleSideSignal TransductionSiteSolid NeoplasmSpecificitySystemTestingTherapeutic InterventionTissuesTranscriptTransgenesTransgenic AnimalsTranslatingTranslational RegulationTranslationsTumor Stem CellsWorkbiological systemscancer stem cellcell typecircadianexperimental studyflygenetic regulatory proteinin vivoknock-downmRNA sequencingnanobodiesnovel strategiesoverexpressionposttranscriptionalribosome profilingsuccesstooltranscriptome sequencingtranslation assay
项目摘要
Project Summary/Abstract
Transcription dominates the gene expression landscape of circadian rhythms and a number of neuroscience
areas. Yet post-transcriptional regulation, including translational regulation and the role of RNA binding
proteins (RBPs), has become increasingly recognized as important in recent years. Moreover, understanding
the roles of RBPs in diverse cell types and diseases and ultimately therapeutic intervention requires identifying
the RNA targets of RBPs. The ribosome can be considered a RBP, so this focus on post-transcriptional
regulation and RBP targets includes identifying ribosome-associated transcripts, namely RNAs that change
their translational status under defined circumstances. RBP identification is particularly challenging from small
numbers of cells, e.g., cancer stem cells within a large, heterogeneous solid tumor or discrete neuronal
subtypes. These settings preclude traditional biochemistry and therefore require new approaches. Two new
methods recently appeared, TRIBE and STAMP, which exploit the RNA editing enzymes ADAR and APOBEC,
respectively. Their ribosome versions, Ribo-STAMP and Ribo-TRIBE, are even more recent and fuse a
ribosomal protein to the editing enzymes. This is so that the enzyme will be near RNAs that are being
translated and will “mark” them by changing their sequence. These edits are identified by mRNA sequencing
and straightforward computational methods, even from single cells. We propose to compare Ribo-STAMP and
Ribo-TRIBE side-by-side in mammalian cell culture systems, to assess their efficacy and to determine the
optimal configuration of ribosome-editing enzyme fusions. We also propose to develop Ribo-TRIBE for use in
Drosophila; we recently discovered that STAMP does not work in this organism, which limits us to TRIBE. We
will then extend these methods to the more biological context of neurons, from Drosophila as well as from
mouse brains. To complement these efforts, we will develop an extension of the TRIBE/STAMP theme called
Nanobody-editing. It consists of fusing the editing enzyme, ADAR or APOBEC, to a GFP-nanobody, which will
then deliver the editing enzyme to any GFP-tagged RNA binding protein or GFP-tagged ribosome. The
chimeric, recombinant protein will be used in vitro as a recombinant protein or expressed in vivo. Nanobody-
editing will facilitate identifying RBP and ribosome targets, because already existing GFP-tagged RBP or
ribosomes can serve as substrates. Moreover, in vitro editing will in many cases obviate the need to generate
new transgenes and transgenic animals. Lastly, TRIBE and as well as Ribo-TRIBE will be used to characterize
translational regulation “around the clock” within a few key Drosophila circadian neurons. These efforts will
deepen our understanding of circadian gene expression regulation as well as provide the community with new
tools with which to study translation in important but challenging biological systems.
项目总结/摘要
转录主导了昼夜节律的基因表达景观和一些神经科学
地区然而,转录后调控,包括翻译调控和RNA结合的作用,
近年来,人们越来越认识到蛋白质(RBP)的重要性。此外,理解
RBPs在不同细胞类型和疾病中的作用以及最终的治疗干预需要确定
RBP的RNA靶点。核糖体可以被认为是一个RBP,所以这个重点是转录后
调节和RBP靶点包括鉴定核糖体相关转录物,即改变
在特定情况下的翻译状态。RBP识别是特别具有挑战性的,
细胞的数量,例如,大的异质性实体瘤或离散的神经元内的癌症干细胞
亚型这些环境排除了传统的生物化学,因此需要新的方法。两个新
最近出现的方法TRIBE和STAMP,它们利用RNA编辑酶阿达尔和APOBEC,
分别它们的核糖体版本Ribo-STAMP和Ribo-TRIBE甚至更近,
核糖体蛋白质到编辑酶。这是为了使酶靠近RNA,
翻译并将通过改变它们的顺序来“标记”它们。这些编辑通过mRNA测序鉴定
和简单的计算方法,甚至从单个细胞。我们建议比较Ribo-STAMP和
在哺乳动物细胞培养系统中并排使用Ribo-TRIBE,以评估它们的功效并确定它们在哺乳动物细胞培养系统中的作用。
核糖体编辑酶融合体的最佳构型。我们还建议开发Ribo-TRIBE,用于
果蝇;我们最近发现STAMP在这种生物体中不起作用,这将我们限制在TRIBE。我们
然后将这些方法扩展到神经元的更多生物学背景,从果蝇以及从
老鼠的大脑为了补充这些努力,我们将开发TRIBE/STAMP主题的扩展,称为
纳米抗体编辑它包括将编辑酶阿达尔或APOBEC融合到GFP纳米抗体上,
然后将编辑酶递送到任何GFP标记的RNA结合蛋白或GFP标记的核糖体。的
嵌合重组蛋白将在体外用作重组蛋白或在体内表达。纳米抗体-
编辑将有助于识别RBP和核糖体靶标,因为已经存在的GFP标记的RBP或
核糖体可以作为底物。此外,在许多情况下,体外编辑将消除产生DNA的需要。
新的转基因和转基因动物。最后,TRIBE和Ribo-TRIBE将用于表征
翻译调控“全天候”在几个关键的果蝇昼夜神经元。这些努力将
加深我们对昼夜节律基因表达调控的理解,并为社区提供新的
在重要但具有挑战性的生物系统中研究翻译的工具。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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MICHAEL ROSBASH其他文献
MICHAEL ROSBASH的其他文献
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{{ truncateString('MICHAEL ROSBASH', 18)}}的其他基金
A new tool for the cell-specific identification of RNA binding protein targets
用于细胞特异性鉴定 RNA 结合蛋白靶标的新工具
- 批准号:
8640299 - 财政年份:2013
- 资助金额:
$ 40.63万 - 项目类别:
A new tool for the cell-specific identification of RNA binding protein targets
用于细胞特异性鉴定 RNA 结合蛋白靶标的新工具
- 批准号:
8735924 - 财政年份:2013
- 资助金额:
$ 40.63万 - 项目类别:
A new tool for the cell-specific identification of RNA binding protein targets
用于细胞特异性鉴定 RNA 结合蛋白靶标的新工具
- 批准号:
9112980 - 财政年份:2013
- 资助金额:
$ 40.63万 - 项目类别:
Addressing protein synthesis regulation within small numbers of discrete neurons
解决少量离散神经元内的蛋白质合成调节问题
- 批准号:
10091418 - 财政年份:2013
- 资助金额:
$ 40.63万 - 项目类别:
A new tool for the cell-specific identification of RNA binding protein targets
用于细胞特异性鉴定 RNA 结合蛋白靶标的新工具
- 批准号:
8913110 - 财政年份:2013
- 资助金额:
$ 40.63万 - 项目类别:
2011 Chronobiology Gordon Research Conference
2011年时间生物学戈登研究会议
- 批准号:
8118328 - 财政年份:2011
- 资助金额:
$ 40.63万 - 项目类别:
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