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

我们提出要解决两个长期存在的问题，在该领域的前mRNA剪接：（1）如何分支 U2的位点识别区（BSRR）是一个单一的独特序列，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）？以及（2）假尿苷如何 （1），这是大量存在于U2 BSRR，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，允许我们用随机化的 BSS序列，在不同的遗传背景下，其中U2中的碱基的数量和组合 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， 在解决上述长期存在的问题方面处于独特地位。提出了三个具体目标。 1.为了扩展由不同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，在前- mRNA也被U2 BSRR识别，我们将用这种5 '- 相邻序列被随机化，在不同的U2（不同的U2）背景下。在这样做时，我们希望 获得BSS及其5'相邻序列的长列表，所述BSS及其5'相邻序列由具有不同 在BSRR中的符号的数量和组合，从而帮助解码符号如何有助于BSS识别。 2.评价U2 BSRR内的BSs是否/如何促进BSS选择/基因表达 有了BSS及其5'端相邻序列，我们将自然地搜索所选序列。 前体mRNA将在U2变体下测试这些前mRNA的剪接效率（不同之处在于 ）。在这样做的时候，我们可以将U2 BSRR中的cytokine与剪接/基因调控联系起来。我们也希望一些U2 变体偏好一个BSS+5'相邻序列而不是另一个，而其他U2变体具有完全的 相同的两个BSS+5'相邻序列之间的相反偏好。我们将创建几个结构 其中两个BSS+5 ′相邻序列被并行放置，并且测试一个BSS相对于另一个BSS的使用 在特定的U2背景下的剪接过程中，从而将β-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 '-邻近序列 由于在任何给定位点处的不完全假尿苷化，存在U2变体的混合物（在碱基上不同）， 细胞我们将详细分析U2变体的混合物如何在特定的BSS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， 从剪接体分离的U2的BSRR，剪接体组装到具有特异性BSS+5'端的前体mRNA上。 相邻序列在这样做的过程中，我们可以评估每个U2变体在自然环境中的行为， 在BSS识别的过程中。一个完整的图片，预计将出现的BSS介导的识别。

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.