Regulation and impact of alternative splicing in biology and disease
选择性剪接在生物学和疾病中的调控和影响
基本信息
- 批准号:10405870
- 负责人:
- 金额:$ 39.25万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2022
- 资助国家:美国
- 起止时间:2022-08-09 至 2027-06-30
- 项目状态:未结题
- 来源:
- 关键词:5&apos Splice SiteAddressAlternative SplicingBiochemicalBioinformaticsBiologicalBiological AssayBiologyCellsCodeDefectDiseaseElementsEssential GenesEventExcisionExonsGene ExpressionGene Expression ProfileGenerationsGenesGenetic DiseasesGenetic TranscriptionGoalsGrowthHeterogeneous-Nuclear RibonucleoproteinsHuman GeneticsHuman GenomeIntronsKineticsKnock-inKnock-outLabelLeadLinkMediatingMessenger RNAMetabolicMethodsModelingMolecularMolecular BiologyMutationOutcomePathway interactionsPositioning AttributeProcessProteinsPublishingRNARNA ProcessingRNA SplicingRegulationReporterRepressionResearchRoleSiteStructureTechniquesTestingTissue DifferentiationTranscriptTranslatingTranslationsU1 Small Nuclear RibonucleoproteinVariantWorkcancer typecell typecis acting elementcombinatorialconformergenetic regulatory proteinhuman diseaseimprovedinsightmRNA PrecursormRNA Stabilitynovelprematureprogramsradioligandreconstitutionsynthetic biologytherapeutic targettissue culturetranscriptomics
项目摘要
Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in
splicing lead to human genetic disease, and splicing mutations in a number of genes involved in growth control
have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing
and splice site recognition are therefore fundamental to understanding regulated gene expression and human
disease. The control of alternative splicing is a highly combinatorial process, where many inputs dictate the
splicing outcome for each exon. A critical feature of these regulatory mechanisms is the specific interaction of
trans-acting splicing factors with cis-acting RNA elements. We use a highly integrated approach to investigate
the molecular mechanisms that regulate pre-mRNA splicing. This includes knockout and knock-in tissue
culture models, reconstitution assays using radioligands, transcriptomics, bioinformatics, kinetics, structure-
function and biochemical techniques. In the next five years, we aim to address several outstanding challenges
in the field, pursuing the following novel research directions. (1) Our demonstration that splicing regulatory
proteins display highly position-dependent activities that negatively or positively influence splice site choice
changed the way we think about the classical splicing activators (SR proteins) and the classical splicing
repressors (hnRNPs). It is now appreciated that the context-dependent activation or repression of U1 snRNP
serves as a gateway to allow the abundant U1 snRNP to fulfill its splicing function and its role to protect the
pre-mRNA from premature degradation. However, it is not understood how splicing regulators achieve
activation or repression of U1snRNP at the 5’ splice site. We aim to dissect the mechanisms of splicing
repression by embracing multi-system approaches and by understanding the role of U1 snRNP conformers in
mediating spliceosomal assembly. (2) Intron retention is an important alternative splicing pathway that has
eluded extensive study. Thus, its regulation is not well-understood. The existence of inefficiently spliced
introns within coding exons (exitrons) further highlights the biological importance of understanding when
introns are removed efficiently and when they are not. We will decipher the rules of efficient intron removal
and investigate the impact of cis-acting elements in this process using synthetic biology approaches. The
argument is that the depth of the sequence variation tested in massively parallel reporter assays is far greater
than the testing landscape that the human genome offers. Here, we will take advantage of our expertise in
experimental molecular biology and bioinformatics. (3) It has become widely appreciated that gene expression
events are highly integrated, with evidence suggesting that most pre-mRNA processing occurs co-
transcriptionally. Defects in any one of these steps has been linked to disease. However, most published
studies evaluate only steady-state levels of gene expression or focus only on a single step. This ignores the
dynamics of gene expression steps that collectively contribute to the generation of proteins from mRNAs.
Thus, it is unclear how the kinetics of RNA processing and mRNA stability translate into an endpoint gene
expression signature. We have established a reliable method to metabolically label nascent RNA, which
allows us to track transcripts from synthesis to degradation. Work in this project will probe how steady state
mRNA levels are established, how the splicing and translation regulator SRSF1 influences mRNA dynamics
and how these processes adjust as a cell undergoes transformation. The goals of our research program are to
obtain a better understanding of exon recognition and alternative splicing. The new mechanistic insights will
be leveraged to improve strategies to therapeutically target this essential gene expression step.
前体mRNA剪接是大多数后生动物基因表达所需的基本过程。缺陷
剪接导致人类遗传疾病,并且在涉及生长控制的许多基因中发生剪接突变
与多种癌症有关了解前体mRNA剪接的基本机制
因此,剪接位点识别是理解调控基因表达和人类
疾病选择性剪接的控制是一个高度组合的过程,其中许多输入决定了选择性剪接。
每个外显子的剪接结果。这些调节机制的一个关键特征是以下物质的特异性相互作用:
具有顺式作用RNA元件的反式作用剪接因子。我们使用高度综合的方法来调查
调节前体mRNA剪接的分子机制。这包括敲除和敲入组织
培养模型,使用放射性配体的重建测定,转录组学,生物信息学,动力学,结构-
功能和生化技术。在未来五年,我们的目标是应对几个突出的挑战
在该领域,追求以下新的研究方向。(1)我们证明了剪接调控
蛋白质显示出高度位置依赖性的活性,
改变了我们对经典剪接激活剂(SR蛋白)和经典剪接的看法,
阻遏物(hnRNP)。现在可以理解,U1 snRNP的上下文依赖性激活或抑制
作为一个网关,允许丰富的U1 snRNP履行其剪接功能,并发挥其保护
前体mRNA过早降解。然而,尚不清楚剪接调节子如何实现
在5'剪接位点激活或抑制U1 snRNP。我们的目标是剖析剪接的机制
通过拥抱多系统方法和理解U1 snRNP构象在抑制中的作用,
介导剪接体组装。(2)内含子保留是重要的选择性剪接途径,
逃避了广泛的研究。因此,它的规则没有得到很好的理解。低效拼接的存在
编码外显子(exitrons)内的内含子进一步强调了理解当
内含子被有效地去除,当它们不被去除时。我们将破译有效去除内含子的规则
并使用合成生物学方法研究顺式作用元件在这一过程中的影响。的
一个论点是,在大规模平行报告基因测定中测试的序列变异的深度要大得多
比人类基因组提供的测试环境更好。在这里,我们将利用我们的专业知识,
实验分子生物学和生物信息学。(3)人们已经广泛认识到,基因表达
事件是高度整合的,有证据表明,大多数前mRNA的加工发生在共同的,
转录。这些步骤中的任何一个缺陷都与疾病有关。然而,大多数出版
研究仅评估基因表达的稳态水平或仅关注单个步骤。这忽略了
基因表达步骤的动力学,这些步骤共同有助于从mRNA产生蛋白质。
因此,目前还不清楚RNA加工的动力学和mRNA稳定性如何转化为终点基因
表达式签名。我们已经建立了一种可靠的方法来代谢标记新生RNA,
让我们可以追踪转录本从合成到降解的全过程在这个项目中,我们将探索稳态
建立mRNA水平,剪接和翻译调节因子SRSF 1如何影响mRNA动力学
以及这些过程如何在细胞发生转化时进行调整。我们研究计划的目标是
更好地理解外显子识别和选择性剪接。新的机械论见解将
可以用来改善治疗靶向这一重要基因表达步骤的策略。
项目成果
期刊论文数量(0)
专著数量(0)
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会议论文数量(0)
专利数量(0)
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Klemens J Hertel其他文献
Klemens J Hertel的其他文献
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{{ truncateString('Klemens J Hertel', 18)}}的其他基金
Regulation and impact of alternative splicing in biology and disease
选择性剪接在生物学和疾病中的调控和影响
- 批准号:
10680397 - 财政年份:2022
- 资助金额:
$ 39.25万 - 项目类别:
Regulation and impact of alternative splicing in biology and disease
选择性剪接在生物学和疾病中的调控和影响
- 批准号:
10833336 - 财政年份:2022
- 资助金额:
$ 39.25万 - 项目类别:
Tracking Gene Expression Dynamics from Transcription to Degradation
跟踪从转录到降解的基因表达动态
- 批准号:
8912925 - 财政年份:2015
- 资助金额:
$ 39.25万 - 项目类别:
The role of alternative pre-mRNA splicing in breast cancer progression
选择性前 mRNA 剪接在乳腺癌进展中的作用
- 批准号:
8322940 - 财政年份:2010
- 资助金额:
$ 39.25万 - 项目类别:
The role of alternative pre-mRNA splicing in breast cancer progression
选择性前 mRNA 剪接在乳腺癌进展中的作用
- 批准号:
7991127 - 财政年份:2010
- 资助金额:
$ 39.25万 - 项目类别:
The role of alternative pre-mRNA splicing in breast cancer progression
选择性前 mRNA 剪接在乳腺癌进展中的作用
- 批准号:
8080450 - 财政年份:2010
- 资助金额:
$ 39.25万 - 项目类别:
Mechanisms of enhancer dependent splice-site activation
增强子依赖性剪接位点激活机制
- 批准号:
7892830 - 财政年份:2009
- 资助金额:
$ 39.25万 - 项目类别:
Genomic Analysis of Alternative Splice-Site Selection
选择性剪接位点选择的基因组分析
- 批准号:
7186157 - 财政年份:2007
- 资助金额:
$ 39.25万 - 项目类别:
Genomic Analysis of Alternative Splice-Site Selection
选择性剪接位点选择的基因组分析
- 批准号:
7383919 - 财政年份:2007
- 资助金额:
$ 39.25万 - 项目类别:
MECHANISMS OF ENHANCER DEPENDENT SPLICE SITE ACTIVATION
增强子依赖性剪接位点激活机制
- 批准号:
6845708 - 财政年份:2001
- 资助金额:
$ 39.25万 - 项目类别:
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