Pseudouridine-mediated branch site recognition/selection during pre-mRNA splicing

我们提出了解决前mRNA剪接领域中的两个长期存在的问题：(1)分支如何 U2的位点识别区(BSRR)是一个单一的唯一序列，通过碱基配对识别 剪接过程中前信使核糖核酸中分支位点序列的变化？和(2)假尿苷是如何 在U2 BSRR中大量存在的(Ψ)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，允许我们以随机的方式筛选前mRNA文库 BSS序列，在不同的遗传背景下，ΨS的数量和组合在U2 BSRR被操纵和控制。我们的初步筛选产生了令人兴奋的初步结果，将我们 以得天独厚的地位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‘相邻序列的列表 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也表明，在BSS前-BSS附近的6个核苷酸序列5‘- MRNA也被U2 BSRR识别，我们将用这个5‘-筛选额外的前mRNA文库 相邻序列是随机的，在不同的U2(不同的Ψ)背景下。通过这样做，我们希望 获得由不同U2变异体选择的BSS及其5‘相邻序列的长列表 ΨS的数字和组合，从而帮助破译ΨS如何对盲源分离识别做出贡献。 2.评估U2 BSRR内的ΨS是否/如何参与血细胞选择/基因表达 有了BSS和ITS 5‘相邻序列，我们将在Natural中搜索选定的序列 发生前信使核糖核酸。这些前mRNAs的剪接效率将在U2变体(不同于 Ψ)。这样，我们就可以将U2BSRR中的Ψ与剪接/基因调控联系起来。我们还预计一些U2 变异体喜欢一个BSS+5‘相邻序列而不是另一个，而其他U2变异体完全 相同的两个BSS+5‘相邻序列之间相反的偏好。我们将创建几个构造 将两个BSS+5‘相邻序列平行放置，并测试其中一个BSS相对于另一个的使用 在特定U2背景下的剪接过程中，从而将Ψ介导的BS选择与替代剪接联系起来。 3.剖析单个U2 RNA变异体如何识别不同的BSS及其5‘-相邻序列 由于在任何给定的位置都存在不完全的假尿路，所以存在U2变异体的混合(Ψ不同)。 细胞。我们将详细分析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在 从剪接体中分离到的U2的BSRR，这些剪接体与特定的BSS+5‘组装到一个前mRNA上。 相邻序列。在这样做的过程中，我们可以评估每个单独的U2变体在自然环境中的表现， 在盲源分离识别的过程中。预计将出现Ψ介导的盲源分离识别的完整图景。

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.