Functional analysis of tRNA splicing endonuclease from Arabidopsis thaliana

tRNA剪接核酸内切酶在一些被内含子中断的tRNA（植物中的tRNA α和tRNA β）的成熟中起重要作用<Tyr><Met>。我们以前已经分离出两个推定的基因编码的催化亚基的tRNA核酸内切酶从拟南芥（AtSen 1和AtSen 2）。为了确定这些基因产物的性质，产生表达它们的转基因拟南芥植物：AtSen 1或AtSen 2 cDNA或这些cDNA中的一种，在每个ORF的156位编码催化三联体的一个氨基酸的His密码子被Ala密码子取代AtSen 1H 156 A和AtSen 2 H156 A）与CaMV 35 S启动子融合，以便在植物细胞中过表达它们。使用农杆菌产生过表达每个基因的植物系。对从这四种不同植物系中分离的总tRNA的北方分析显示，<Met>仅在AtSen 1H 156 A植物中积累了显著水平的含内含子的前体tRNA。这一发现表明 ...更多信息 AtSen 1本身（而不是AtSen 2）具有在5'和3'剪接位点切割tRNA的能力。为了证实这一结论，我们比较了gusA mRNA中琥珀密码子通过在<Met>这些转基因植物和野生型植物的愈伤组织中瞬时表达的抑制子tRNA β 1的翻译抑制效率。正如所料，仅在AtSen 1H 156 A植物中观察到相对较低的GUS活性，这可能是因为在共表达的gusA mRNA中琥珀密码子的翻译抑制减少。用Ni-NTA树脂纯化这些蛋白。在体外剪接测定的^&lt;32？P标记的模板（携带凸出-螺旋-凸出基序（8 H8）的pNtY 9 * T7 M1）是在AtSen 1或AtSen 2存在下完成的。该结果表明，每个催化亚基AtSen 1或AtSen 2可以独立地切割人工植物前体tRNA的BHB结构中的5'和3'剪接位点<Tyr>。然而，典型的含内含子的前tRNA（即，环-螺旋-凸起基序）在其中一个亚基或两个亚基存在下是不良底物，这可能意味着即使在植物前体tRNA剪接中也需要与酵母Sen 54 p和/或Sen 15 p同源的额外亚基。这些假定亚基的分离正在进行中。少

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