Splicing Regulation of Spinal Muscular Atrophy Genes

摘要：人类有两个几乎相同的生存运动神经元（SMN）基因，SMN 1和SMN 2。 由于SMN 1缺失和/或突变导致的低SMN水平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性疾病。SMN 2无法补偿SMN 1的损失， 外显子7的跳跃。由于两种基因的全长mRNA编码相同的蛋白质，因此SMN 2的校正 外显子7剪接提供了最好的治疗选择之一。我们发现了内含子剪接沉默器N1（ISS- N1）作为反义寡核苷酸（阿索）介导的SMN 2纠正的有希望的治疗靶点 外显子7剪接。Nusinersen（SpinrazaTM）是一种ISS-N1靶向阿索，最近获得FDA（美国）批准 作为治疗SMA的第一种药物。虽然nusinersen成功地阻止了大量 大多数SMA婴儿的速度和疾病进展缓慢，但问题仍然存在， 复苏的程度。因此，迫切需要制定替代和/或补充机制， 基础疗法，以改善SMA的治疗。这一建议旨在了解转录- SMN基因的偶联剪接调控（TCSR），以发现增强SMN的新治疗靶点 SMN 2的水平。这个项目是基于这样的前提，即转录起始和延伸调节两者 SMN 2转录水平和SMN 2外显子7剪接。我们提出的研究将结合联合收割机的几个互补 和强大的技术，包括天然延伸转录测序（NET-seq）， 连续测序（PRO-seq）、体内结构探测和沉积在膜上的复合物的亲和纯化。 在RNA聚合酶II（pol II）的转录过程中新生RNA。为确保研究的可行性， 已经产生了SMN 2“超级小基因”，由全长SMN 2启动子、所有外显子及其侧翼组成 SMN 2的内含子序列和3 ′非翻译区（3 ′ UTR）。我们提出这项研究是鉴于 SMN基因产生大量环状RNA（cRNA或circRNA）的相关发现。在目标1中， 我们将检验SMN 2启动子和转录区中的顺式元件 调节转录（起始和延伸），并因此影响外显子7是否将被包括或 跳过。利用PRO-seq技术，我们将分析SMN在不同细胞中转录过程中的pol II暂停位点 类型我们将确定转录暂停位点是否参与SMN 2外显子7的TCSR。我们将评估 小分子（包括转录和剪接调节剂）对SMN 2外显子7 TCSR的影响。我们将 还确定转录和剪接调节剂是否影响SMN cRNA的产生。我们将分析 组织，以确定SMN产生的cRNA是否与SMA相关 发病机制利用SMN 2超小基因文库，我们将揭示启动子元件在SMN 2基因表达中的作用。 SMN 2外显子7的剪接。我们还将检查SMN 2内特定位点的DNA甲基化是否有任何影响， SMN 2外显子7的TCSR。在目标2中，我们将检验以下假设： 转录调节SMN 2转录水平和SMN 2外显子7剪接。使用SMN 2超级小基因， 我们将筛选参与SMN 2外显子7 TCSR的启动子相关因子。我们最近将雇用 建立了基于亲和力的技术来鉴定和表征与转录相关的蛋白质 延伸复合物（EC）在近端启动子以及基因体和终止处募集 SMN 2的网站。我们将通过质谱分析EC。我们将验证已确定因素的作用， SMN 2外显子7的TCSR，通过过表达和耗尽它们。我们将确定这些与EC相关的 因子在SMA小鼠模型的组织中异常表达。我们亦会研究 在SMN 2外显子7的TCSR中新发现的含有α样序列的长链非编码RNA（IncRNA）。在 目的3，我们将检验RNA结构参与SMN 2外显子7的TCSR的假设。的区域 将基于通过PRO-seq数据鉴定的pol II暂停位点来选择参与相关结构形成的pol 和Mfold程序的计算机预测。我们将验证结构的作用， SMN 2超小基因。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之间的任何协调相互作用的潜在影响， SMN 2外显子7 TCSR结构。我们还将研究DHX 9（一种RNA解旋酶）在 SMN 2外显子7的TCSR和SMN cRNA的生物发生。我们将进行一项有限的研究， 前四个cRNA的功能。我们将检查这些cRNA的缺失是否对SMN有任何影响。 转录，SMN外显子7剪接和SMN水平。 整体转录组和蛋白质组。研究结果将揭示新的治疗靶点，以改善和/或 SMA的补充疗法。结果将促进我们对SMN 2基因调控的理解， 鉴定SMA新疾病调节剂。

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.