Splicing Regulation of Spinal Muscular Atrophy Genes

脊髓性肌萎缩症基因的剪接调控

基本信息

批准号：
10596591
负责人：
RAVINDRA N SINGH
金额：
$ 33.47万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-07-06 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10596591
关键词：
3&apos Untranslated Regions Affect Affinity Affinity Chromatography Alu-Like Repetitive Sequence Antisense Oligonucleotides Biogenesis Cessation of life Code Compensation Complementary therapies Complex Coupled DNA Methylation Data Deposition Disease Disease Progression Elements Ensure Exons FDA approved Gene Expression Regulation Generations Genes Genetic Genetic Diseases Genetic Transcription Goals Human In Vitro Infant Infant Mortality Lead Length Libraries Mass Spectrum Analysis Mediating Messenger RNA Molecular Motor Neurons Mutation Nuclear Outcome Pathogenesis Pharmacotherapy Polymerase Promoter Regions Proteins Proteome RNA RNA Helicase RNA Polymerase II RNA Splicing Recovery Regulation Regulatory Element Role Running SMN1 gene SMN2 gene Site Speed Spinal Muscular Atrophy Structure Techniques Testing Therapeutic Tissues Transact Transcript Transcription Elongation Transcription Initiation Untranslated RNA cell type circular RNA exon skipping experimental study improved in silico in vivo mouse model new therapeutic target novel overexpression programs promoter recruit small molecule survival motor neuron gene therapeutic target transcriptome

项目摘要

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.

摘要：人类有两个几乎相同的运动神经元生存 (SMN) 基因副本：SMN1 和 SMN2。 SMN1 缺失和/或突变导致 SMN 水平低，导致脊髓性肌萎缩症 (SMA)，这是一种主要的疾病与婴儿死亡率相关的遗传病无法弥补 SMN1 造成的损失。跳过外显子 7。由于两个基因的全长 mRNA 编码相同的蛋白质，因此需要修正 SMN2 我们发现内含子剪接沉默器 N1 (ISS-) 是最好的治疗选择之一。 N1) 作为反义寡核苷酸 (ASO) 介导的 SMN2 校正的有前景的治疗靶点 Nusinersen (SpinrazaTM) 是一种针对 ISS-N1 的 ASO，最近获得 FDA（美国）的批准。作为第一个治疗 SMA 的药物，nusinersen 已成功阻止大量死亡。大多数 SMA 婴儿并减缓疾病进展，但速度和速度方面的问题仍然存在因此，迫切需要开发替代和/或补充机制。该提案旨在了解转录-改善 SMA 的治疗。 SMN 基因的耦合剪接调节 (TCSR) 以发现增强 SMN 的治疗新靶点该项目基于转录起始和延伸调节两者的前提。 SMN2 转录水平和 SMN2 外显子 7 剪接我们提出的研究将结合几个互补的研究。和强大的技术，包括天然延伸转录测序（NET-seq）、精密核连续测序 (PRO-seq)、体内结构探测和沉积在复合物上的复合物的亲和纯化 RNA 聚合酶 II (pol II) 转录过程中的新生 RNA。生成了 SMN2“超级小基因”，由全长 SMN2 启动子、所有外显子及其侧翼组成我们根据SMN2的内含子序列和3￠非翻译区(3￠UTR)提出这项研究。相关发现是 SMN 基因产生大量环状 RNA（cRNA 或 circRNA）。我们将测试 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 是否被包含或使用 PRO-seq，我们将分析不同细胞中 SMN 转录过程中的 pol II 暂停位点。我们将确定 SMN2 外显子 7 的 TCSR 中是否涉及转录暂停位点。我们将评估小分子（包括转录和剪接调节剂）对 SMN2 外显子 7 的 TCSR 的影响。我们将还确定转录和剪接调节剂是否影响 SMN cRNA 的生成。 SMA 小鼠模型的组织以确定 SMN 生成的 cRNA 是否与 SMA 相关利用 SMN2 超级小基因文库，我们将揭示启动子元件在发病机制中的作用。 SMN2 外显子 7 的剪接。我们还将检查 SMN2 内特定位点的 DNA 甲基化是否有任何影响关于 SMN2 外显子 7 的 TCSR。在目标 2 中，我们将检验以下假设：在转录调节 SMN2 转录水平和 SMN2 外显子 7 剪接使用 SMN2 超级小基因，我们将筛选参与SMN2外显子7的TCSR的启动子相关因子。我们最近将采用建立基于亲和力的技术来识别和表征与转录相关的蛋白质在近端启动子以及基因体和终止处募集的延伸复合物（EC）我们将通过质谱分析 ECs 我们将验证已识别因素的作用。通过过度表达和耗尽 SMN2 外显子 7 的 TCSR 我们将确定这些 EC 是否相关。我们还将研究 SMA 小鼠模型组织中异常表达的因子。新发现的长非编码 RNA (IncRNA) 在 SMN2 外显子 7 的 TCSR 中含有 Alu 样序列。目标 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 数据识别的 pol II 暂停位点选择参与相关结构形成的以及 Mfold 程序的计算机预测，我们将验证结构在环境中的作用。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（一种 RNA 解旋酶）在 SMN2 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来揭示潜力。我们将检查这些 cRNA 的消耗是否对 SMN 有影响。转录、SMN 外显子 7 剪接和 SMN 水平。我们还将确定耗尽对整体转录组和蛋白质组的研究结果将揭示改进和/或改善的新治疗靶点。 SMA 的补充疗法将增进我们对 SMN2 基因调控的理解。确定 SMA 的新疾病调节剂。

项目成果

期刊论文数量（45）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Unfolding the mystery of alternative splicing through a unique method of in vivo selection.

通过独特的体内选择方法揭开选择性剪接的神秘面纱。

DOI：
发表时间：
2007-05-01
期刊：
影响因子：
0
作者：
Singh; Ravindra N
通讯作者：
Ravindra N

A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes.

多外显子跳跃检测分析揭示了脊髓性肌萎缩症基因剪接亚型的惊人多样性。

DOI：
发表时间：
2012
期刊：
影响因子：
3.7
作者：
Singh, Natalia N;Seo, Joonbae;Rahn, Sarah J;Singh, Ravindra N
通讯作者：
Singh, Ravindra N

Splicing regulation in spinal muscular atrophy by an RNA structure formed by long-distance interactions.

长距离相互作用形成的RNA结构对脊髓性肌萎缩症的剪接调节。

DOI：
发表时间：
2015-04
期刊：
影响因子：
5.2
作者：
Singh, Natalia N;Lee, Brian M;Singh, Ravindra N
通讯作者：
Singh, Ravindra N

Severe impairment of male reproductive organ development in a low SMN expressing mouse model of spinal muscular atrophy.

低 SMN 表达的脊髓性肌萎缩小鼠模型中雄性生殖器官发育严重受损。

DOI：
发表时间：
2016-02-02
期刊：
影响因子：
4.6
作者：
Ottesen, Eric W;Howell, Matthew D;Singh, Natalia N;Seo, Joonbae;Whitley, Elizabeth M;Singh, Ravindra N
通讯作者：
Singh, Ravindra N

Activation of a cryptic 5' splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene.

神秘 5 剪接位点的激活可逆转脊髓性肌萎缩症基因中致病性剪接位点突变的影响。