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：
YI-TAO YU
金额：
$ 33.04万
依托单位：
UNIVERSITY OF ROCHESTER
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

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.

我们建议在前MRNA剪接领域解决两个长期存在的问题：（1）分支如何 U2的位点识别区（BSRR）是一个独特的序列，识别（通过碱基对）剪接过程中MRNA中的各种分支位点序列（BSS）？（2）伪岛如何（ψ）在U2 BSRR中绝对存在，这有助于这种识别？要解决这些问题，我们最近开发了一个筛选系统，使我们能够筛选一个带有随机的MRNA库 BSS序列，在各种遗传背景下，其中U2中的数量和组合 BSRR被操纵和控制。我们的初始屏幕产生了令人兴奋的初步结果以独特的位置解决近似问题。提出了三个具体目标。 1。扩展BS的列表及其5'相邻序列，以不同的U2变体识别我们将基于初步结果，并继续筛选库（随机BSS）。鉴于我们在我们的初步实验中还显示了BSS中BSS的6-核苷酸序列5'-序列 mRNA也被U2 BSRR认可，我们将使用此5'-筛选前MRNA的其他库在不同的U2（ψ）背景下，相邻的序列是随机的。这样，我们希望获得一长串BS及其5'相邻序列，由不同的U2变体选择不同 BSRR中ψs的数字和组合，从而有助于解码ψs如何对BSS识别贡献。 2。评估U2 BSRR内是否有助于BSS选择 /基因表达借助BSS及其5'相邻序列，我们将自然搜索所选序列发生前MRNA。这些前MRNA的剪接效率将在U2变体下进行测试（不同 ψ）。这样，我们可以将U2 BSRR中的ψ与剪接/基因调节联系起来。我们还希望有一些U2 变体偏爱一个BSS+5'相邻序列，而不是另一个BSS+5'，而其他U2变体完全具有相同的两个BS+5'相邻序列之间的偏好相反。我们将创建几个构造将两个BS+5'相邻序列并联放置，并测试一个BSS在另一个BS上的使用在特定U2背景下的剪接过程中，将ψ介导的BS选择与替代剪接联系起来。 3。剖析单个U2 RNA变体如何识别各种BSS及其5'-ADJACEST序列由于在任何给定位点的假性不完全，因此存在U2变体的混合物（ψ不同）细胞。我们将详细阐述U2变体的混合物如何单独识别特定的BSS。剪接。我们将使用牛津纳米孔技术在单分子水平上定量映射ψ 从剪接体中分离出的U2的BSRR，它们与特定的BSS+5'组合到Pre-MRNA上相邻序列。通过这样做，我们可以评估每个U2变体在自然背景下的行为在BSS识别过程中。预计ψ介导的BSS识别的完整图片将出现。