Functional analysis of tRNA splicing endonuclease from Arabidopsis thaliana

拟南芥 tRNA 剪接核酸内切酶的功能分析

• 批准号: 13680766
• 负责人: AKAMA Kazuhito
• 金额: $ 2.3万
• 依托单位: Shimane University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680766/
• 关键词: tRNA splicing; tRNA splicing endonuclease; intoron; Arabidopsis; transgenic plant; amber suppressor tRNA; Agrobacterium; in vitro transcription; translation; サプレッサーtRNA; バルジ・ヘリックス・バルジ; トランスジェニク植物

tRNA splicing endonuclease plays an important role in the maturation of some tRNAs (tRNA^<Tyr> and tRNA^<Met> in plants) that are interrupted by introns. We have previously isolated two putative genes coding for catalytic subunits of tRNA endonucleases from Arabidopsis (AtSen1 and AtSen2). To determine the properties of the products of these genes, transgenic Arabidopsis plants expressing them were produced : AtSen1 or AtSen2 cDNA or one of these cDNAs with substitution of a His codon encoding one amino acid of the catalytic triad by an Ala codon in position 156 in each ORF (designated AtSen1H156A and AtSen2H156A) were fused to the CaMV 35S promoter in order to overexpress them in plant cells. Plant lines overexpressing each gene were produced using Agrobacterium. Northern analysis of the total tRNAs isolated from these four different plant lines revealed that a significant level of intron-containing pre-tRNA^<Met> was accumulated only in AtSen1H156A plants. This finding suggests that … More AtSen1 by itself (but not AtSen2) has the ability to cleave the tRNA at the 5' and 3' splice sites. In order to confirm this conclusion, we compared the efficiency of the translational suppression of an amber codon in gusA mRNA via suppressor tRNA^<Met> transiently expressed in callus tissues from these transgenic plants and wild-type plants. As expected, relatively lower GUS activity was observed only in AtSen1H156A plants, probably because of a decrease in the translational suppression of the amber codon in the co-expressed gusA mRNA.In parallel, protein synthesis (AtSen1 and AtSen2) was performed in vitro using the RTS kit (Roche). These proteins were purified with the Ni-NTA resin. In vitro splicing assay of ^<32?P labeled template (pNtY9*T7M1 that carries bulge-helix-bulge motif (8H8)) was done in the presence of AtSen1 or AtSen2. This result indicated that each catalytic subunit, AtSen1 or AtSen2, can independently cleave both 5' and 3' splice sites in the BHB structure of artificial plant pre-tRNA^<Tyr>. However, a typical intron-containing pre-tRNA (i.e., loop-helix-bulge motif) seen in plants was poor substrates in the presence of one of the subunits or both of them, which may imply requirement of additional subunits homologous to yeast Sen54p and/or Sen15p even in plant pre-tRNA splicing. Isolation of these putative subunits is now in progress. Less

TRNA剪接内切酶在一些被内含子打断的tRNAs(植物中的tRNA^&lt；Tyr&gt；和tRNA^&lt；Met&gt；)的成熟过程中起着重要的作用。我们之前已经从拟南芥中分离到了两个编码tRNA内切酶催化亚基的基因(AtSen1和AtSen2)。为了确定这些基因产物的性质，获得了表达这些基因的转基因拟南芥植株：AtSen1或AtSen2，或者其中的一个，在每个ORF的156位用Ala密码子取代编码催化三联体的一个氨基酸的His密码子(分别命名为AtSen1H156A和AtSen2H156A)与CaMV 35S启动子融合，以便在植物细胞中过表达。利用农杆菌技术获得了高表达每个基因的植株系。Northern分析表明，仅在AtSen1H156A植株中积累了大量含有前tRNA^&lt；Met&gt；的内含子。这一发现表明，…更多的AtSen1(而不是AtSen2)本身具有切割5‘和3’剪接位点的tRNA的能力。为了证实这一结论，我们比较了在这些转基因植株和野生型植株的愈伤组织组织中瞬时表达的tRNA^&lt；Met&gt；对Gusa mRNA中琥珀色密码子的翻译抑制效率。正如预期的那样，只有在AtSen1H156A植物中观察到相对较低的GUS活性，这可能是因为共表达的Gusa mRNA中琥珀密码子的翻译抑制减少。同时，蛋白质合成(AtSen1和AtSen2)是使用RTS试剂盒(罗氏)进行的。用Ni-NTA树脂对这些蛋白进行纯化。在AtSen1或AtSen2存在下，进行了~(32)P标记模板(携带凸起-螺旋-凸起基序(8H8)的pNtY9*T7M1)的体外剪接实验。这一结果表明，每个催化亚基AtSen1或AtSen2都可以独立地切割人工植物Pre-tRNA^&lt；Tyr&gt；的BHB结构中的5‘和3’剪接位点。然而，在植物中看到的典型的含有内含子的前tRNA(即环-螺旋-凸起基序)在其中一个亚基或两者都存在的情况下是糟糕的底物，这可能意味着即使在植物前tRNA剪接中也需要与酵母Sen54p和/或Sen15p同源的额外亚基。分离这些假定的亚基目前正在进行中。较少

项目成果

Akama K, Beier H: "Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli."Nucleic Acid Research. 31(4). 1197-1207 (2003)

Akama K、Beier H：“翻译无义密码子抑制作为转化拟南芥下胚轴衍生愈伤组织中功能性前 tRNA 剪接的指标。”核酸研究。

Y.Yukawa, H.Fan, K.Akama, H.Beier, H.J.Gross, M.Sugiura: "A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors"The Plant Journal. 28・5. 583-594 (2001)

Y.Yukawa、H.Fan、K.Akama、H.Beier、H.J.Gross、M.Sugiura：“支持植物内含子 tRNA 前体转录、加工、剪接和修饰的烟草核提取物”《植物杂志》28・5.583-594 (2001)

Akama K, Beier H: "Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli."Nucleic Acid Research. 31. 1197-1207 (2003)

Akama K、Beier H：“翻译无义密码子抑制作为转化拟南芥下胚轴衍生愈伤组织中功能性前 tRNA 剪接的指标。”核酸研究。

Yukawa Y, Fan H, Akama K, Beier H, Gross HJ, Sugiura M: "A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors."Plant Journal. 28(5). 583-594 (2001)

Yukawa Y、Fan H、Akama K、Beier H、Gross HJ、Sugiura M：“支持含植物内含子 tRNA 前体转录、加工、剪接和修饰的烟草核提取物。”《植物杂志》。

Yukawa Y, Fan H, Akama K, Beier H, Gross HJ, Sugiura M: "A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors."Plant Journal. 28. 583-594 (2001)

Yukawa Y、Fan H、Akama K、Beier H、Gross HJ、Sugiura M：“支持含植物内含子 tRNA 前体转录、加工、剪接和修饰的烟草核提取物。”《植物杂志》。