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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。