Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation
细胞质加帽与转录后基因调控的关系
基本信息
- 批准号:7888807
- 负责人:
- 金额:$ 30.5万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2010
- 资助国家:美国
- 起止时间:2010-04-01 至 2014-03-31
- 项目状态:已结题
- 来源:
- 关键词:AbbreviationsActive SitesAddressArabidopsisBiochemicalBiologicalBiological ProcessCell ExtractsCellsCellular StressCleaved cellCommitComplexCytoplasmCytoplasmic GranulesDevelopmentDiphosphatesDiseaseDominant-Negative MutationEmbryonic DevelopmentEnzymesErythroid CellsExcisionFamilyGene ExpressionGene Expression ProfileGene Expression RegulationGeneticGenetic TranslationGlobinGlycerolGrowth and Development functionLeadLearningLinkMalignant NeoplasmsMammalian CellMass Spectrum AnalysisMemoryMessenger RNAMolecular GeneticsMultienzyme ComplexesMutationNeuronsNeurosciencesNuclearPhosphotransferasesPlayPolyadenylationPopulationPost-Transcriptional RegulationProcessProductionProtein BindingProteinsProteomePublishingRNARNA InterferenceRNA SplicingRecoveryRegulationRoleSiteSmall RNAStem cellsStressStructureSystemThinkingTranslatingTranslation InitiationTranslationsWorkendonucleasemRNA DecaymRNA Surveillancenovelpublic health relevanceresearch studyresponserestorationtool
项目摘要
DESCRIPTION (provided by applicant): The addition of a cap to the 5' end of all eukaryotic mRNAs is the first step in post-transcriptional processing, and its removal is generally thought to irreversibly commit mRNA to decay. In erythroid cells nonsense- containing 2-globin mRNA is cleaved by a cytoplasmic endonuclease to generate decay intermediates that are both stable and capped. Although most capping enzyme is nuclear, we identified a 140 kDa cytoplasmic capping enzyme complex that contains a 5'-monophosphate kinase capable of transforming the 5' end of decapped RNA into a diphosphate capping substrate. Although cytoplasmic capping enzyme is not associated with either P bodies or stress granules evidence for its biological role was demonstrated by the reduced recovery from stress of cells expressing a dominant negative form of this protein. The corollary to cytoplasmic capping is an uncapped transcriptome, evidence of which was recently identified by our lab in mammalian cells and by others in Arabidopsis. These mRNAs were linked to cytoplasmic capping by their increased representation in the uncapped pool following expression of a dominant negative form of capping enzyme. Aim 1 will use biochemical approaches to identify and characterize the components of the cytoplasmic capping enzyme complex, with particular emphasis on the novel 5'-monophosphate kinase. These findings will guide development of molecular and genetic tools for characterizing the biological function of cytoplasmic capping. Experiments in Aim 2 will characterize the 5' ends of a selected number of the identified re-capping substrates and study dynamic changes in their cap status after interfering with cytoplasmic capping. The 3' ends of these RNAs will also be examined to determine if deadenylation and/or oligouridylylation lead to the accumulation of uncapped mRNAs. The last portion of Aim 2 will combine deep sequencing with the tools developed in Aim 1 to generate a comprehensive picture of the uncapped transcriptome and its relationship to cytoplasmic capping. Aim 3 will address the biological relevance of cytoplasmic capping as it relates to the cycling of mRNAs between translating and non-translating states. These experiments will examine the impact of altering the size and number of P bodies and interfering with different steps leading to decapping and P body assembly, and examine the relationship of microRNA silencing to the accumulation of uncapped mRNAs and/or their restoration to the translating pool. Lastly, iTRAQ mass spectrometry will be used to determine if altering cytoplasmic capping changes the complexity of the proteome. Cytoplasmic capping has the potential to broadly impact our understanding of normal and disease processes that are linked to post-transcriptional control, including stem cells, embryonic development, cancer and neuroscience.
PUBLIC HEALTH RELEVANCE: The endpoint of most gene expression is the production of a protein product, and the regulation of mRNA translation is essential for such diverse processes as development, learning and memory, the cellular response to stress and the development and growth of cancers. Non-translating mRNAs are stored for later use or degraded, and little is known about the state of these stored mRNAs or how they are re-activated. This proposal examines cytoplasmic capping as a new regulatory process with broad implications for post- transcriptional gene regulation, RNA silencing and translational control.
描述(由申请人提供):在所有真核mRNA的5'末端添加帽子是转录后处理的第一步,通常认为它的去除是不可逆地将mRNA腐烂的。在红细胞中,含有2-珠蛋白mRNA的胡说八道被细胞质内核酸酶切割,以产生稳定和封盖的衰减中间体。尽管大多数上限酶都是核的,但我们确定了140 kDa的细胞质上限酶复合物,该酶络合物含有5'单磷酸酯激酶,能够将Decapped RNA的5'末端转化为双磷酸盐上限底物。尽管细胞质上限酶与P体或应激颗粒的证据无关,证明其生物学作用是通过从表达该蛋白质的主要负形式的细胞的压力中恢复的恢复来证明的。细胞质封盖的推论是一个未覆盖的转录组,我们的实验室最近在哺乳动物细胞中鉴定出其证据,在哺乳动物细胞中和其他拟南芥中鉴定出来。这些mRNA与细胞质上限相关联,其在表达封闭酶的主要负面形式后,它们在未封闭的池中增加的表示。 AIM 1将使用生化方法来识别和表征细胞质上限酶复合物的成分,并特别强调了新型的5'-单磷酸激酶。这些发现将指导分子和遗传工具的开发,以表征细胞质上限的生物学功能。 AIM 2中的实验将表征所选数量的重新封闭底物的5端,并研究干扰细胞质上限后其帽状态的动态变化。还将检查这些RNA的3'末端,以确定去烯基化和/或寡烯基化是否会导致非限制的mRNA的积累。 AIM 2的最后一部分将与AIM 1中开发的工具相结合,以产生无盖转录组及其与细胞质封盖的关系的全面图片。 AIM 3将解决与翻译和非翻译状态之间mRNA循环有关的细胞质封盖的生物学相关性。这些实验将检查改变p身体的大小和数量并干扰导致decapping和p身体组装的不同步骤的影响,并检查microRNA沉默与无上限mRNA的积累和/或它们与翻译池的累积的关系。最后,将使用ITRAQ质谱法来确定改变细胞质封盖是否会改变蛋白质组的复杂性。细胞质封盖有可能广泛影响我们对与转录后控制有关的正常和疾病过程的理解,包括干细胞,胚胎发育,癌症和神经科学。
公共卫生相关性:大多数基因表达的终点是蛋白质产物的产生,而mRNA翻译的调节对于诸如发展,学习和记忆,对压力的细胞反应以及癌症的发展和生长等多种过程至关重要。非翻译mRNA存储以供以后使用或降级,对这些存储的mRNA的状态或如何重新激活它们几乎不知情。该建议将细胞质封盖视为一种新的调节过程,对转录后基因调控,RNA沉默和翻译控制具有广泛的影响。
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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DANIEL R. SCHOENBERG其他文献
DANIEL R. SCHOENBERG的其他文献
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{{ truncateString('DANIEL R. SCHOENBERG', 18)}}的其他基金
Relationship of cytoplasmic capping to post-transcriptional gene regulation
细胞质加帽与转录后基因调控的关系
- 批准号:
9249712 - 财政年份:2010
- 资助金额:
$ 30.5万 - 项目类别:
Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation
细胞质加帽与转录后基因调控的关系
- 批准号:
8445319 - 财政年份:2010
- 资助金额:
$ 30.5万 - 项目类别:
Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation
细胞质加帽与转录后基因调控的关系
- 批准号:
8040924 - 财政年份:2010
- 资助金额:
$ 30.5万 - 项目类别:
Relationship of cytoplasmic capping to post-transcriptional gene regulation
细胞质加帽与转录后基因调控的关系
- 批准号:
9118224 - 财政年份:2010
- 资助金额:
$ 30.5万 - 项目类别:
Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation
细胞质加帽与转录后基因调控的关系
- 批准号:
8242018 - 财政年份:2010
- 资助金额:
$ 30.5万 - 项目类别:
Nonsense codon activation of endonuclease-mediated mRNA decay
核酸内切酶介导的 mRNA 衰变的无义密码子激活
- 批准号:
8208188 - 财政年份:2009
- 资助金额:
$ 30.5万 - 项目类别:
Nonsense codon activation of endonuclease-mediated mRNA decay
核酸内切酶介导的 mRNA 衰变的无义密码子激活
- 批准号:
7751927 - 财政年份:2009
- 资助金额:
$ 30.5万 - 项目类别:
Nonsense codon activation of endonuclease-mediated mRNA decay
核酸内切酶介导的 mRNA 衰变的无义密码子激活
- 批准号:
8004999 - 财政年份:2009
- 资助金额:
$ 30.5万 - 项目类别:
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