Pseudouridine-mediated branch site recognition/selection during pre-mRNA splicing
前 mRNA 剪接过程中假尿苷介导的分支位点识别/选择
基本信息
- 批准号:10612829
- 负责人:
- 金额:$ 33.04万
- 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:2020-08-10 至 2025-04-30
- 项目状态:未结题
- 来源:
- 关键词:AddressAffectAlternative SplicingBase PairingBase SequenceBioinformaticsCellsCodeCoupledDataDiseaseGene ExpressionGene Expression RegulationGenesGeneticIncidenceIndividualLibrariesLinkMapsMediatingModificationNucleotidesPlayPositioning AttributeProcessPseudouridineQuantitative Reverse Transcriptase PCRRNARNA SplicingRandomizedRegulationReporterRoleScreening ResultSiteSpliceosomesSystemTechnologyTestingTimeU2 small nuclear RNAVariantVertebratesWorkXenopusXenopus oocyteYeastsclinically relevantexperimental studymRNA Precursornanoporepreferencescreeningsingle molecule
项目摘要
We propose to solve two long-standing problems in the field of pre-mRNA splicing: (1) how does the branch
site recognition region (BSRR) of U2, which is a single unique sequence, recognize (via base-pairing) a
variety of branch site sequences (BSS) in pre-mRNAs during splicing? And (2) how does the pseudouridine
(Ψ), which is abundantly present in the U2 BSRR, contribute to this recognition? To address these questions,
we recently developed a screening system, allowing us to screen a library of pre-mRNAs with randomized
BSS sequences, under various genetic backgrounds where the numbers and combinations of Ψs in the U2
BSRR are manipulated and controlled. Our initial screen has generated exciting preliminary results, placing us
in a unique position to solve the aforementioned long-standing problems. Three specific aims are proposed.
1. To expand the list of BSS and its 5' adjacent sequences recognized by different U2 variants
We will build on our preliminary results and continue to screen the library (randomized BSS). Given that we
have also shown in our preliminary experiments that a 6-nucleotide sequence 5'-adjacent to the BSS in pre-
mRNA is also recognized by the U2 BSRR, we will screen additional libraries of pre-mRNAs with this 5'-
adjacent sequence being randomized, under different U2 (differ in Ψ) backgrounds. In doing so, we expect to
obtain a long list of BSS and its 5' adjacent sequences selected by different U2 variants with different
numbers and combinations of Ψs in the BSRR, thus helping decode how Ψs contribute to BSS recognition.
2. To evaluate whether/how Ψs within the U2 BSRR contribute to BSS selection / gene expression
With the BSS and its 5' adjacent sequences handy, we will search for the selected sequences in naturally
occurring pre-mRNAs. The splicing efficiency of these pre-mRNAs will be tested under U2 variants (differ in
Ψ). In doing so, we can link Ψ in the U2 BSRR to splicing/gene regulation. We also expect that some U2
variants prefer one BSS+5' adjacent sequence over another, whereas other U2 variants have completely
opposite preference between the same two BSS+5' adjacent sequences. We will create several constructs
with two BSS+5' adjacent sequences being placed in parallel, and test the usage of one BSS over the other
during splicing under specific U2 backgrounds, thus linking Ψ-mediated BSS selection to alternative splicing.
3. To dissect how individual U2 RNA variants recognize various BSSs and their 5'-adjacent sequences
Due to incomplete pseudouridylation at any given site, there exists a mixture of U2 variants (differ in Ψ) in
cells. We will dissect, in detail, how the mixture of U2 variants individually recognizes a specific BSS during
splicing. We will use the Oxford nanopore technology to quantitatively map, at single molecule level, Ψ in the
BSRR of U2 isolated from the spliceosomes, which are assembled onto a pre-mRNA with a specific BSS+5'
adjacent sequence. In doing so, we can assess how each individual U2 variant behaves, in a natural context,
in the process of BSS recognition. A complete picture of Ψ-mediated BSS recognition is expected to emerge.
我们提出解决pre-mRNA剪接领域两个长期存在的问题:(1)分支如何进行
U2 的位点识别区域 (BSRR) 是一个独特的序列,可识别(通过碱基配对)
剪接过程中前体 mRNA 中的各种分支位点序列 (BSS)? (2) 假尿苷如何
U2 BSRR 中大量存在的 (Ψ) 是否有助于这种认识?为了解决这些问题,
我们最近开发了一个筛选系统,使我们能够用随机筛选方法筛选前体 mRNA 库。
BSS序列,在不同的遗传背景下,U2中Ψ的数量和组合
BSRR 是可操纵和控制的。我们的初始筛选产生了令人兴奋的初步结果,使我们
具有独特的地位来解决上述长期存在的问题。提出了三个具体目标。
1. 扩展不同U2变体识别的BSS及其5'相邻序列列表
我们将在初步结果的基础上继续筛选文库(随机 BSS)。鉴于我们
在我们的初步实验中还表明,在预-中与 BSS 5'-相邻的 6 个核苷酸序列
mRNA 也被 U2 BSRR 识别,我们将用这个 5'- 筛选额外的前体 mRNA 文库
在不同的 U2(Ψ 不同)背景下,相邻序列被随机化。在此过程中,我们期望
获得一长串 BSS 及其由具有不同 U2 变体选择的 5' 相邻序列
BSRR 中 Ψ 的数量和组合,从而帮助解码 Ψ 如何有助于 BSS 识别。
2. 评估 U2 BSRR 内的 Ψ 是否/如何有助于 BSS 选择/基因表达
有了 BSS 及其 5' 相邻序列,我们将自然地搜索所选序列
发生前 mRNA。这些 pre-mRNA 的剪接效率将在 U2 变体(不同
Ψ)。通过这样做,我们可以将 U2 BSRR 中的 Ψ 与剪接/基因调控联系起来。我们还预计一些 U2
变体更喜欢一个 BSS+5' 相邻序列而不是另一个,而其他 U2 变体完全具有
相同的两个BSS+5'相邻序列之间的偏好相反。我们将创建几个结构
将两个 BSS+5' 相邻序列并行放置,并测试一个 BSS 相对于另一个 BSS 的使用情况
在特定 U2 背景下的剪接过程中,从而将 Ψ 介导的 BSS 选择与选择性剪接联系起来。
3. 剖析单个 U2 RNA 变体如何识别各种 BSS 及其 5'-相邻序列
由于任何给定位点的假尿苷化不完全,因此存在 U2 变体的混合物(Ψ 不同)
细胞。我们将详细剖析 U2 变体的混合物如何在过程中单独识别特定的 BSS
拼接。我们将使用牛津纳米孔技术在单分子水平上定量绘制 Ψ
从剪接体中分离出的 U2 的 BSRR,剪接体组装到具有特定 BSS+5' 的前 mRNA 上
相邻序列。通过这样做,我们可以评估每个 U2 变体在自然环境中的行为,
在BSS识别过程中。 Ψ 介导的 BSS 识别的完整图像有望出现。
项目成果
期刊论文数量(6)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies.
- DOI:10.3390/biomedicines9050550
- 发表时间:2021-05-14
- 期刊:
- 影响因子:4.7
- 作者:Adachi H;Hengesbach M;Yu YT;Morais P
- 通讯作者:Morais P
Spliceosomal snRNA Epitranscriptomics.
- DOI:10.3389/fgene.2021.652129
- 发表时间:2021
- 期刊:
- 影响因子:3.7
- 作者:Morais P;Adachi H;Yu YT
- 通讯作者:Yu YT
The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines.
- DOI:10.3389/fcell.2021.789427
- 发表时间:2021
- 期刊:
- 影响因子:5.5
- 作者:Morais P;Adachi H;Yu YT
- 通讯作者:Yu YT
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{{ truncateString('YI-TAO YU', 18)}}的其他基金
Pseudouridine-mediated branch site recognition/selection during pre-mRNA splicing
前 mRNA 剪接过程中假尿苷介导的分支位点识别/选择
- 批准号:
10383755 - 财政年份:2020
- 资助金额:
$ 33.04万 - 项目类别:
Pseudouridine-mediated branch site recognition/selection during pre-mRNA splicing
前 mRNA 剪接过程中假尿苷介导的分支位点识别/选择
- 批准号:
10231213 - 财政年份:2020
- 资助金额:
$ 33.04万 - 项目类别:
Pseudouridine-mediated branch site recognition/selection during pre-mRNA splicing
前 mRNA 剪接过程中假尿苷介导的分支位点识别/选择
- 批准号:
10026485 - 财政年份:2020
- 资助金额:
$ 33.04万 - 项目类别:
Suppression of disease causingnonsense mutations by targeted mRNA pseudouridylation
通过靶向 mRNA 假尿苷化抑制引起无义突变的疾病
- 批准号:
9805188 - 财政年份:2019
- 资助金额:
$ 33.04万 - 项目类别:
Expanding the genetic code by targeted pseudouridylation
通过靶向假尿苷化扩展遗传密码
- 批准号:
8550118 - 财政年份:2012
- 资助金额:
$ 33.04万 - 项目类别:
Expanding the genetic code by targeted pseudouridylation
通过靶向假尿苷化扩展遗传密码
- 批准号:
8419298 - 财政年份:2012
- 资助金额:
$ 33.04万 - 项目类别:
Regulation of telomerase activity and aging by 2'-O-methylation in telomerase RNA
端粒酶RNA中2-O-甲基化对端粒酶活性和衰老的调节
- 批准号:
8516934 - 财政年份:2012
- 资助金额:
$ 33.04万 - 项目类别:
Expanding the genetic code by targeted pseudouridylation
通过靶向假尿苷化扩展遗传密码
- 批准号:
8914641 - 财政年份:2012
- 资助金额:
$ 33.04万 - 项目类别:
Expanding the genetic code by targeted pseudouridylation
通过靶向假尿苷化扩展遗传密码
- 批准号:
8720024 - 财政年份:2012
- 资助金额:
$ 33.04万 - 项目类别:
Regulation of telomerase activity and aging by 2'-O-methylation in telomerase RNA
端粒酶RNA中2-O-甲基化对端粒酶活性和衰老的调节
- 批准号:
8242209 - 财政年份:2012
- 资助金额:
$ 33.04万 - 项目类别:
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