Structural basis of IRE1 mRNA splicing mechanism

IRE1 mRNA剪接机制的结构基础

• 批准号: 18570106
• 负责人: KOJIMA Chojiro
• 金额: $ 2.57万
• 依托单位: Nara Institute of Science and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570106/
• 关键词: Structural Biology; NMR; Signal Transduction; Protein; Molecular Recognition; 耕造生物学

IRE1 on endoplasmic reticulum (ER) is a transmembrane protein (type I) with N- and C-terminal domains in lumen and cytoplasm sides, respectively, and the N-terminal domain senses the accumulation of denatured proteins. Activated Irelp specifically cleaves mRNA of transcription activation factor HAC1 of the endoplasmic reticulum chaperon genes, and enables translation of Haclp by performing splicing without spliceosome. This new splicing has characteristics such as (1) 5' and 3' sides are cleaved independently, (2) tRNA ligase is involved, (3) it is performed in cytoplasm, and it is the signal transduction mechanism which is widely conserved from yeast to human. Here, we focused on the elucidation of the structural bases of IRE1 pathway in ER stress response, in particular the splicing mechanism without spliceosome.The structural studies of Ire1p RNase domain, C-terminal domain and the stem loop of HAC1 mRNA including cleavage site are performed by NMR and crystallization. The major results are the following. (1) For NMR experiments, the Irelp RNase domain is enriched with ^13C and ^15N, and the sample conditions including buffers and temperature are optimized. The best HSQC spectrum is obtained with weak alkaline HEPES buffer. (2) The expression and purification of the Irelp C-terminal domain are successfully performed, and subjected to crystallization with the aid of Professor Toshio Hakoshima (NAIST) . The crystals are obtained but they are not suitable for structure analysis. (3) Two stem loops of HAC1 mRNA including cleavage site are used for the structural study using NMR. Imino proton signals indicate the presence of the base pair not only in stem regions but also in loop regions. (4) The variants of functionally important residues are prepared and used to study the RNase activities and the structural changes. (5) NMR titration experiments are performed to identify the interface residues between the Irelp RNase domain and the stem loop of HAC1 mRNA.

内质网（endoplasmic reticulum， ER）上的IRE1是一种跨膜蛋白（type I），其N端和c端结构域分别位于管腔侧和细胞质侧，N端结构域感知变性蛋白的积累。激活的Irelp特异性切割内质网伴侣基因转录激活因子HAC1的mRNA，并通过无剪接体的剪接实现Haclp的翻译。这种新的剪接具有：(1)5′和3′侧独立切割，(2)涉及tRNA连接酶，(3)在细胞质中进行，是一种从酵母到人类广泛保守的信号转导机制。本文重点阐述了内质网应激反应中IRE1通路的结构基础，特别是无剪接体的剪接机制。利用核磁共振和结晶技术对Ire1p核糖核酸结构域、c端结构域和HAC1 mRNA茎环（包括裂解位点）进行了结构研究。主要结果如下。(1)在核磁共振实验中，Irelp RNase结构域富集了^13C和^15N，并优化了缓冲液和温度等样品条件。弱碱性HEPES缓冲液获得最佳HSQC谱。(2)在Toshio Hakoshima教授（NAIST）的帮助下，成功地表达和纯化了Irelp c -末端结构域，并进行了结晶。得到了晶体，但不适合进行结构分析。(3)采用核磁共振技术对HAC1 mRNA的两个茎环（包括裂解位点）进行结构研究。微质子信号表明该碱基对不仅存在于茎区，也存在于环区。(4)制备了功能重要残基的变体，并用于研究RNase活性和结构变化。(5)通过核磁共振滴定实验，鉴定了Irelp RNase结构域与HAC1 mRNA茎环之间的界面残基。

项目成果

NMR spectroscopic study of a DNA duplex with mercury-mediated T-T base pairs

• DOI: 10.1080/15257770600686154
• 发表时间: 2006-01-01
• 期刊: NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS
• 影响因子: 1.3
• 作者: Tanaka, Yoshiyuki;Yamaguchi, Hiroshi;Ono, Akira
• 通讯作者: Ono, Akira

Structure of membrane proteins

膜蛋白的结构

• 发表时间: 2008
• 期刊: Structural Biology: Introduction and Background
• 作者: Tahara;Y.;G. Masunaga;H. Ota.;田原 義太慶;田原義太慶・増永元・太田英利;田原義太慶・増永元・太田英利;太田英利;太田英利・高橋亮雄;太田英利;C. Kojima
• 通讯作者: C. Kojima

Enzymatic Characterization of 5-Methlthioribose 1-Phosphate Isomerase from Bacillus subtilis

枯草芽孢杆菌 5-甲基硫核糖 1-磷酸异构酶的酶学表征

• 发表时间: 2007
• 期刊: Biosci. Biotectnol. Biochem. 71
• 作者: Tahara;Y.;G. Masunaga;H. Ota.;田原 義太慶;田原義太慶・増永元・太田英利;田原義太慶・増永元・太田英利;太田英利;太田英利・高橋亮雄;太田英利;C. Kojima;Y. Saito
• 通讯作者: Y. Saito

Structural analysis of phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Natronomonas pharaonis 趋光传感器蛋白 HtrII 连接区的结构分析

• 发表时间: 2007
• 期刊: Biochemistry 46
• 作者: Tahara;Y.;G. Masunaga;H. Ota.;田原 義太慶;田原義太慶・増永元・太田英利;田原義太慶・増永元・太田英利;太田英利;太田英利・高橋亮雄;太田英利;C. Kojima;Y. Saito;K. Hayashi
• 通讯作者: K. Hayashi

1H, 13C and 15N resonance assignments of the VAP-A: OSBP complex

VAP-A 的 1H、13C 和 15N 共振分配：OSBP 复合物

• DOI: 10.1007/s10858-006-9057-2
• 发表时间: 2006
• 期刊: Journal of Biomolecular NMR
• 影响因子: 2.7
• 作者: Kyoko Furuita;M. Mishima;C. Kojima
• 通讯作者: C. Kojima