Molecular mechanism of mammalian mRNA surveillance

哺乳动物 mRNA 监测的分子机制

基本信息

批准号：
17209010
负责人：
OHNO Shigeo
金额：
$ 32.86万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17209010/
关键词：
nonsense mutation mRNA degradation mRNA surveillance P53 protein kinase PIK remated protein kinase RNA helicase ATPase

项目摘要

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNA containing premature termination codons (PTCs). In mammalian cells, recognition of PTCs requires translation and depends on the presence on the mRNA with the splicing-dependent exon junction complex (EJC). While it is known that a key event in the triggering of NMD is phosphorylation of the trans-acting factor, Upf1, by SMG-1, the relationship between Upf1 phosphorylation and PTC recognition remains undetermined. Here we show that SMG-1 binds to the mRNA-associated components of the EJC, Upf2, Upf3b, elF4A3, Magoh, and Y14. Further, we describe a novel complex that contains the NMD factors SMG-1 and Upf1, and the translation termination release factors eRF1 and eRF3 (SURF). Importantly, an association between SURF and the EJC is required for SMG-1-mediated Upf1 phosphorylation and NMD. Thus, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link betw … More een the EJC and recognition of PTCs and triggers NMD.Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression ineukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition tothe unique serine/threonine protein kinase (PIKK) domain that is different from classical proteinkinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused onone of the PIKK members, SMG-1 that is involved in RNA surveillance termed nonsense-mediatedmRNA decay (N MD), to analyze the roles of conserved and SMG-1 specific sequences on the intrinsickinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intactcells revealed that the long N-terminal region and the conserved leucine in the FATC domain wereessential for SMG-1 kinase activity. However, the conserved tryptophan in the TS domain and theFATC domain was not. In addition, the long insertion region between PIKK and FATC domains wasnot essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e.,the distantly located N-terminal and C-terminal sequences were essential for the intrinsic kinaseactivity.In order to confirm our previous results using SMG-1 inhibitors, we evaluated the effects of NMD inhibition by siRNA-mediated knockdown of SMG-1 or Upf1 on the phenotype of Ullrich disease, an autosomal recessive congenital muscular dystrophy. The patient studied showed a homozygousframeshift mutation with a PTC in the collagen VI A2 gene, which encodes a truncated but partiallyfunctional protein. The patient's fibroblasts showed a nearly complete loss of the triple-helical collagenVI protein and functional defects in the extracellular matrix (ECM) due to the crucial deficiency of the collagen VI a2 protein. We have shown that siRNA-mediated knockdown of SMG-1 or Upf1 causes theupregulation of the mutant triple-helical collagen VI, resulting in the formation of partially functionalECM. We suggest that the inhibition of NMD may be useful as a therapeutic approach to treat somehuman genetic diseases exacerbated by NMD. Less

废话介导的mRNA衰变（NMD）是一种监视机制，可降解含有过早终止密码子（PTC）的mRNA。在哺乳动物细胞中，对PTC的识别需要翻译，并取决于依赖剪接的外显子连接复合物（EJC）上的mRNA上的存在。尽管众所周知，NMD触发的关键事件是通过SMG-1的跨作用因子UPF1的磷酸化，但UPF1磷酸化与PTC识别之间的关系仍然不确定。在这里，我们表明SMG-1与EJC，UPF2，UPF3B，ELF4A3，MAGOH和Y14的MRNA相关组件结合。此外，我们描述了包含NMD因子SMG-1和UPF1的新型复合物，以及翻译终止释放因子ERF1和ERF3（SURF）。重要的是，SMG-1介导的UPF1磷酸化和NMD需要冲浪与EJC之间的关联。 That, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link betw … More een the EJC and recognition of PTCs and triggers NMD.Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in基因表达无核细胞的监测。他们参与了负责基因组稳定性，mRNA质量和翻译的机制。除了独特的丝氨酸/苏氨酸蛋白激酶（Pikk）结构域外，它们共享一个大型N末端结构域和C端FATC结构域，该结构域与经典蛋白氨基酶不同。但是，架子的结构功能关系尚不清楚。在这里，我们集中了Pikk成员的SMG-1，其中涉及称为废话介导的mRNA衰变（N MD）的RNA监测，以分析保守和SMG-1特异性序列在intrinsickinase活性上的作用。对纯化系统和完整赛中SMG-1的点和缺失突变体的分析表明，SMG-1激酶活性的FATC结构域中长的N末端区域和保守的亮氨酸是必不可少的。但是，TS结构域和FATC结构域中的保守色氨酸不是。另外，Pikk和FATC结构域之间的长插入区域对于SMG-1激酶活性并不是必需的。这些结果表明SMG-1的意外特征，即，异常定位的N末端和C末端序列对于内在的激酶活性至关重要。为了使用SMG-1抑制剂确认我们先前的结果，我们评估了NMD抑制作用通过Sirna介导的SMG-1或UPF1的抑制作用对ull smg-1或Ullrich soson的抑制作用的影响营养不良。研究菌病的患者在胶原蛋白VI A2基因中显示出纯合丝避变突变，该突变编码截短但功能性的蛋白质。由于胶原蛋白VI A2蛋白的关键缺陷，患者的成纤维细胞几乎完全丧失了三螺旋胶原VI蛋白和细胞外基质（ECM）的功能缺陷。我们已经表明，siRNA介导的SMG-1或UPF1的敲低导致突变三螺旋胶原VI的upe缩，从而形成了部分功能。我们建议，NMD的抑制作用可能是一种治疗NMD加剧的人口遗传疾病的治疗方法。较少的