Cross-regulation between transcription and pre-mRNA splicing

摘要/项目摘要 真核生物中普遍存在的转录和前mRNA加工需要了解 它们同时发生并相互调节的机制。我们的长期目标是 确定共转录内含子去除的机制以及共转录剪接如何 有助于基因表达调控。在之前的资助下，我的实验室开创了 纯化和测序新生RNA的策略-转录和RNA的瞬时中间体 处理-相对于延伸RNA聚合酶II（Pol II）的位置定量前mRNA剪接， 基因大多数内含子从Pol II中出现后就会被去除，这表明Pol II和剪接体 被定时一起行动并且在物理上彼此靠近。了解到这一点，我们的总体目标是：（i） 阐明了“全或无”RNA加工的共转录机制，其中个体新生的 转录物或者在3'端完全剪接和切割（全部），或者完全未剪接和未切割（无）。我的实验室 最近通过对全长新生RNA进行测序发现了这一点，它是一种主要的共转录调控因子， B-珠蛋白基因表达的机制。(ii)确定拼接的可能性如何受到限制， 转录继续进行并且新生转录物变得更长。以及（iii）确定其命运和可能的功能 未经处理的记录核心假设是新生RNA结合阳性-和 负作用RNA结合蛋白和/或进行分子间碱基配对和压缩增加 因为新生链在转录过程中生长。我们将通过追求三个具体目标来检验这一假设：1） 开发一种方法来确定新生RNA的局部碱基配对行为，使用DMS化学， 新生RNA纯化。这一目标的结果将告诉我们新生RNA在多大程度上成为 在转录过程中瞬时结构化，如果局部结构与对转录的积极或消极影响相关， 剪接，2）鉴定在剪接过程中在生长的新生链上积累的激活蛋白和/或阻遏蛋白， 转录，使用生物化学纯化和体内标记方法。3)确定核反应 未处理的“无”成绩单是否延误处理？或者他们会被降级吗？为此，我们 已经建立了A-count方法，该方法从5'端到3'端对整个转录本进行测序，包括 polyA尾的全长，其将通过核苷酸类型和数量计数。我们的初步结果 显示核多聚腺苷酸结合蛋白PABPN 1在有丝分裂期间受磷酸化调节， 表明具有内含子的核保留mRNA的不同命运。PABPN 1是一种蛋白质突变， 眼咽肌营养不良症（OPMD）。这项研究意义重大，因为这些机制 在前mRNA加工水平上操作可以改变蛋白质产物的量至少10倍，表明 RNA加工对基因表达的重要性不亚于转录因子的调节。

Abstract/Project Summary The ubiquitous need for transcription and pre-mRNA processing in eukaryotes requires an understanding of the mechanisms by which they occur simultaneously and regulate each other. Our long-term goals are to determine the mechanisms that govern co-transcriptional intron removal and how co-transcriptional splicing contributes to gene expression regulation. With previous support from this grant, my lab has pioneered strategies for purifying and sequencing nascent RNA – the transient intermediates in transcription and RNA processing – to quantify pre-mRNA splicing relative to the position of elongating RNA Polymerase II (Pol II) on the gene. Most introns are removed as soon as they emerge from Pol II, indicating that Pol II and the spliceosome are timed to act together and physically close to one another. Knowing this, our overall objectives are to (i) elucidate the co-transcriptional mechanism of “all-or-none” RNA processing, in which individual nascent transcripts are either fully spliced and cleaved at the 3’ end (all) or fully unspliced and uncleaved (none). My lab discovered this recently by sequencing full-length nascent RNAs, and it is a major co-transcriptional regulatory mechanism for b-globin gene expression. (ii) Determine how the potential for splicing becomes limited as transcription proceeds and the nascent transcript gets longer. And (iii) Determine the fate and possible function of the unprocessed transcripts. The central hypothesis is that the potential for nascent RNA to bind positive- and negative-acting RNA binding proteins and/or to undergo intermolecular base-pairing and compaction increases as the nascent chain grows during transcription. We will test this hypothesis by pursuing three specific aims: 1) develop a method to determine the local base-pairing behavior of nascent RNA, using DMS chemistry and nascent RNA purification. The results of this aim will tell us the degree to which nascent RNA becomes transiently structured during transcription and if local structures correlate with positive or negative effects on splicing, 2) identify activator and/or repressor proteins that accumulate on the growing nascent chain during transcription, using biochemical purification and in vivo labeling approaches. 3) determine the nuclear response to unprocessed “none” transcripts. Are they delayed in processing? Or will they be degraded? For this aim, we have established the A-count method, which sequences the entire transcript from 5’ end to 3’ end, including the whole length of the polyA tail, which will be counted by nucleotide type and number. Our preliminary results show that the nuclear polyA binding protein PABPN1 is regulated by phosphorylation during mitosis, suggesting differential fates for nuclear retained mRNAs with introns. PABPN1 is a protein mutated in Oculopharyngeal muscular dystrophy (OPMD). The proposed research is significant because these mechanisms operating at the level of pre-mRNA processing can change protein product amounts by at least 10-fold, showing that RNA processing is as important for gene expression as regulation by transcription factors.