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
基本信息
- 批准号:10507429
- 负责人:
- 金额:$ 42.9万
- 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:2022-08-12 至 2024-07-31
- 项目状态:已结题
- 来源:
- 关键词:5&apos Splice SiteAffinityAntibodiesBindingBinding SitesBiological AssayCellsChromatinComplexDataDisease modelEnvironmentFractionationFrequenciesGene ExpressionGeneticGenomeGoalsHumanImmunoprecipitationIntronsMapsMedical GeneticsMethodsMutationNuclearPathologicPatientsPlayPolyadenylationProceduresProcessProteinsRNARNA SplicingRNase protection assayReactionRibonucleasesRoleSiteSystemTissuesU1 Small Nuclear RibonucleoproteinU2 Small Nuclear RibonucleoproteinVariantcell typecrosslinkgenetic regulatory proteinhuman diseasein vivoinsightmRNA Precursorprematuretooltranscriptome
项目摘要
PROJECT SUMMARY/ABSTRACT
We propose to develop new methods for the global identification of protein and RNP interactions with nascent
unspliced RNA. We will use these methods to produce comprehensive maps of spliceosomal U1 snRNP binding
across the human transcriptome. U1 functions in pre-mRNA splicing, in the suppression of premature
cleavage/polyadenylation, and in the nuclear retention of RNA. However, information on its binding sites is very
limited, and how these sites differ for the different functions of U1 is not understood. Unspliced introns in nascent
RNA fractionate with the chromatin, and we recently showed that within the chromatin compartment, splicing
factors engage in interactions not seen elsewhere. We developed methods to isolate proteins and RNP’s that
allowed identification of new regulatory proteins interacting with the U2 snRNP, and the isolation of U2-bound
pre-mRNA fragments encompassing the intronic branch points of HEK293 cells. We call this method
fractionation/immunopurification/RNAse protection (FIRP) and find the FIRP maps of U2/pre-mRNA interactions
to be more comprehensive than previous approaches for mapping branchpoints. We now propose to adapt FIRP
to characterizing interactions of the U1 snRNP with pre-mRNA. We will optimize the extraction of material from
chromatin to obtain U1 snRNP complexes bound to pre-mRNA. We will develop new anti-U1 antibodies that
allow 5’ splice site binding analysis across all types of cells. These methods will be applied both to FIRP assays
of U1 snRNP binding and to iCLIP analyses of U1 protein contacts on chromatin-associated RNA. By
characterizing U1 binding sites on a global scale and developing methods for assaying its interactions in different
cells, regulatory environments and genetic backgrounds, we can examine the processes of 5’ splice site
recognition with new breadth and precision.
项目摘要/摘要
我们建议开发新的方法来全球鉴定蛋白质和RNP与新生的相互作用
未剪接的RNA。我们将使用这些方法来制作剪接体U1 SnRNP结合的全面图谱
在人类转录组中。U1在前-mRNA剪接中的作用,在抑制早产中的作用
裂解/多聚腺苷化,以及在RNA的核保留中。然而,有关其结合位点的信息非常多。
有限,而且这些位点对于U1的不同功能有何不同尚不清楚。新生中的未剪接内含子
RNA与染色质分离,我们最近发现在染色质隔间内,剪接
各种因素相互作用,这在其他地方是看不到的。我们开发了分离蛋白质和RNP的方法
允许鉴定与U2 SnRNP相互作用的新调节蛋白,并分离U2结合蛋白
HEK293细胞内含子分支点的前-mRNA片段。我们称此方法为
分级/免疫纯化/核糖核酸酶保护(FIRP)和寻找U2/Pre-mRNA相互作用的FIRP图谱
比以前的绘制分支点的方法更全面。我们现在建议修改FIRP
目的:研究U1SnRNP与Pre-mRNA的相互作用。我们将优化从
染色质获得与Pre-mRNA结合的U1SnRNP复合体。我们将开发新的抗U1抗体,
允许对所有类型的细胞进行5‘剪接位点结合分析。这些方法都将应用于FIRP分析
并对染色质相关RNA上的U1蛋白接触进行了iCLIP分析。通过
全球范围内U1结合位点的特征及其在不同物种中相互作用的分析方法
细胞、调控环境和遗传背景,我们可以研究5‘剪接位点的过程
识别具有新的广度和精确度。
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
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:
$ 42.9万 - 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:
$ 42.9万 - 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:
$ 42.9万 - 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:
10589873 - 财政年份:2020
- 资助金额:
$ 42.9万 - 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:
9898152 - 财政年份:2018
- 资助金额:
$ 42.9万 - 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:
$ 42.9万 - 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:
$ 42.9万 - 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:
$ 42.9万 - 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:
$ 42.9万 - 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:
$ 42.9万 - 项目类别:
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