Splicing regulation of spinal muscular atrophy genes

摘要：人类有两个几乎相同的存活运动神经元(SMN)基因，SMN1和SMN2。 由于SMN1缺失和/或突变导致的SMN水平低下导致脊髓性肌萎缩症(SMA)，这是一种主要的 与婴儿死亡率相关的遗传病。由于以下原因，SMN1无法补偿SMN1的损失 跳过外显子7。由于两个基因的全长mRNAs编码相同的蛋白质，因此SMN2的校正 外显子7剪接提供了最佳的治疗选择之一。我们发现了内含子剪接消音器N1(ISS- N1)作为反义寡核苷酸(ASO)介导的SMN2校正的有希望的治疗靶点 外显子7剪接。Nusinesen(SpinrazaTM)是一种针对ISS-N1的ASO，最近获得了FDA(美国)的批准 作为治疗SMA的首选药物。虽然努斯纳森已经成功地阻止了大量 大多数SMA婴儿并延缓了疾病的进展，但在速度和速度方面的问题仍然存在 恢复的程度。因此，迫切需要制定替代和/或补充机制-- 用于改善SMA治疗的基础疗法。这项建议旨在理解抄写- SMN基因的偶联剪接调控(TCSR)发现增强SMN的新治疗靶点 来自SM2的水平。这个项目的前提是转录起始和伸长都受到调控 SMN 2转录水平和SMN 2外显子7剪接。我们提议的研究将结合几个互补的 和强大的技术，包括自然延长转录测序(NET-SEQ)，精确核 连续测序、体内结构探测和亲和纯化 RNA聚合酶II(PolII)转录过程中的新生RNA。为确保研究的可行性，我们 已经产生了一个由全长SMN2启动子、所有外显子及其侧翼组成的SMN2超微型基因 内含子序列和SMN2的3？非翻译区(3？非翻译区)。我们根据以下情况提出这项研究 SMN基因产生大量环状RNA(CRNAs或CircRNAs)的相关发现。在目标1中， 我们将检验SMN2启动子和转录区域内的顺式元件的假设 调节转录(起始和延伸)，从而影响外显子7是否将被包括或 已跳过。利用Pro-seq，我们将分析不同细胞中smn转录过程中polII的停顿位置。 类型。我们将确定转录暂停位点是否参与SMN2外显子7的TCSR。我们将评估 小分子，包括转录和剪接调节子，对SMN2外显子7的TCSR的影响。我们将 还要确定转录和剪接调节剂是否影响SMN CRNAs的产生。我们将分析 从SMA小鼠模型获取组织以确定SMN产生的CRNAs是否与SMA相关 发病机制。利用SMN2超微型基因文库，我们将揭示启动子元件在 SMN 2外显子7的剪接。我们还将检查SMN内特定位点的DNA甲基化是否有任何影响 在目标2中，我们将检验这样一种假设，即特定的因子在过程中招募 转录调控SMN 2转录水平和SMN 2外显子7剪接。使用SMN2超微型燃气轮机， 我们将筛选与SMN2外显子7的TCSR相关的启动子相关因子。 建立了基于亲和力的技术来识别和表征与转录相关的蛋白质 在启动子近端以及基因体和末端招募的延伸复合体(ECs) SMN2的位置。我们将用质谱学方法分析ECs。我们将验证已确定的因素在 Smn2基因外显子7的TCSR过度表达和缺失。我们将确定是否有任何这些与EC相关的 因子在SMA模型小鼠的组织中异常表达。我们还将研究 新发现的SMN2第7外显子TCSR中含有类Alu序列的长非编码RNA(IncRNAs)。 目的3，我们将检验RNA结构参与SMN2外显子7的TCSR的假设。 将根据PRO-SEQ数据确定的POLII停顿地点选择参与相关结构形成的人员 以及通过Mold程序进行的电子预测。我们将验证结构在 SMN2超微型稀土。我们将通过体内和体外结构探测来确认RNA结构的存在。 我们将测试启动子区域和RNA之间任何协调相互作用的潜在影响 Smn2外显子7的TCSR结构。我们还将研究DHX9，一个RNA解旋酶，在 SMN基因外显子7的TCSR与SMN CRNAs的生物发生我们将进行一项有限的研究以发现潜在的 排名前四的CRNAs的功能。我们将研究这些CRNA的耗尽是否会对SMN产生任何影响 转录，SMN外显子7剪接和SMN水平。我们还将确定枯竭对 总转录组和蛋白质组。研究结果将揭示新的治疗目标，以改善和/或 SMA的补充疗法。这一结果将促进我们对SMN2基因调控和 确定SMA的新疾病修饰物。

Summary: Humans have two nearly identical copies of Survival Motor Neuron (SMN) gene, SMN1 and SMN2. Low SMN levels due to deletion and/or mutation of SMN1 lead to spinal muscular atrophy (SMA), a major genetic disease associated with infant mortality. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7. Since the full-length mRNAs of both genes code for identical proteins, correction of SMN2 exon 7 splicing provides one of the best therapeutic options. We discovered Intronic Splicing Silencer N1 (ISS- N1) as a promising therapeutic target for an antisense oligonucleotide (ASO)-mediated correction of SMN2 exon 7 splicing. Nusinersen (SpinrazaTM), an ISS-N1-targeting ASO, was recently approved by the FDA (USA) as the first drug for the treatment of SMA. While nusinersen has been successful in halting deaths of a vast majority of SMA infants and slow the disease progression, problems still persist with respect to the speed and extent of recovery. Hence there is an urgent need to develop alternative and/or complementary mechanism- based therapies for an improved treatment of SMA. This proposal is aimed at understanding the transcription- coupled splicing regulation (TCSR) of the SMN genes to uncover novel therapeutic targets for enhancing SMN levels from SMN2. This project is based on the premise that transcription initiation and elongation regulate both SMN2 transcripts levels and SMN2 exon 7 splicing. Our proposed study will combine several complementary and powerful techniques, including native elongating transcription sequencing (NET-seq), precision nuclear run-on sequencing (PRO-seq), in vivo structure probing and affinity purification of complexes deposited on the nascent RNAs during transcription by RNA polymerase II (pol II). To ensure the feasibility of our study, we have generated a SMN2 “super minigene” comprised of the full-length SMN2 promoter, all exons, their flanking intronic sequences and the 3¢ untranslated region (3¢UTR) of SMN2. We are proposing this study in the light of a related discovery that SMN genes produce a vast repertoire of circular RNAs (cRNAs or circRNAs). In Aim 1, we will test the hypothesis that cis-elements within both the promoter and the transcribed region of SMN2 regulate transcription (initiation and elongation) and consequently influence whether exon 7 will be included or skipped. Employing PRO-seq, we will analyze pol II pause sites during transcription of SMN in different cell types. We will determine if transcription pause sites are involved in TCSR of SMN2 exon 7. We will assess the effect of small molecules, including transcription and splicing modulators, on TCSR of SMN2 exon 7. We will also determine if transcription and splicing modulators affect the generation of SMN cRNAs. We will analyze tissues from SMA mouse models to determine if cRNAs generated by SMN have relevance to SMA pathogenesis. Employing a library of SMN2 super minigenes, we will uncover the role of promoter elements in splicing of SMN2 exon 7. We will also examine if DNA methylation of specific sites within SMN2 has any effect on TCSR of SMN2 exon 7. In Aim 2, we will test the hypothesis that specific factors that are recruited during transcription regulate both SMN2 transcript levels and SMN2 exon 7 splicing. Using the SMN2 super minigene, we will screen for promoter-associated factors involved in TCSR of SMN2 exon 7. We will employ recently established affinity-based techniques to identify and characterize proteins associated with the transcription elongation complexes (ECs) recruited at the proximal promoter as well as at the gene body and termination sites of SMN2. We will analyze ECs by mass spectroscopy. We will validate the role of the identified factors in TCSR of SMN2 exon 7 by overexpressing and depleting them. We will determine if any of these EC-associated factors are aberrantly expressed in tissues of mouse models of SMA. We will also examine the role of the newly identified long non-coding RNAs (IncRNAs) harboring Alu-like sequences in TCSR of SMN2 exon 7. In Aim 3, we will test the hypothesis that RNA structures is involved in TCSR of SMN2 exon 7. Regions that are involved in relevant structure formation will be selected based on pol II pause sites identified by PRO-seq data and on in silico prediction by the Mfold program. We will validate the role of structures in the context of the SMN2 super minigene. We will confirm the presence of RNA structures by in vivo and in vitro structure probing. We will test a potential impact of any coordinated interactions between the promoter region and the RNA structure on TCSR of SMN2 exon 7. We will also examine the potential role of DHX9, an RNA helicase, in TCSR of SMN2 exon 7 and biogenesis of SMN cRNAs. We will perform a limited study to uncover potential functions of the top four cRNAs. We will examine if the depletion of these cRNAs has any effect on SMN transcription, SMN exon 7 splicing and SMN levels.We will also determine the effect of the depletion on the overall transcriptome and proteome. Findings will reveal novel therapeutic targets for improved and/or complementary therapies for SMA. Outcome will advance our understanding of SMN2 gene regulation and identify novel disease modifiers of SMA.