Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts
Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts
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DOI：
10.1023/a:1006486315360
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发表时间：
2000-09-01
影响因子：
5.1
通讯作者：
Beier, H
中科院分区：
文献类型：
--
作者：
Akama, K;Junker, V;Beier, H
含内含子的tRNA基因在核植物基因组中是例外的。似乎只有编码tRNA（GPsiA）（Tyr）和延伸tRNA（M）et（C）mAU的两个tRNA基因家族含有间插序列。我们先前已经研究了小麦胚芽剪接核酸内切酶所需的功能，有效和准确的内含子切除拟南芥前tRNA（酪氨酸）。在这里，我们研究了拟南芥延长tRNA（Met）基因在两种不同来源的植物提取物中的表达。该基因首先在HeLa或烟草细胞核提取物中转录，然后在小麦胚芽S23提取物和烟草系统中检查含内含子的tRNA（Met）前体的剪接。结果表明，在两种植物提取物中，前tRNA（Met）转化为成熟tRNA非常有效。为了阐明特定核苷酸在3'和5'剪接位点的潜在作用，以及在任一提取物中用于前tRNA（Met）剪接的结构化内含子的潜在作用，我们通过突变分析进行了系统调查。结果表明，胞苷残基在内含子-外显子边界损害前tRNA（Met）的剪接和一个高度结构化的内含子是必不可少的前tRNA（Met）的剪接。具有三到四个碱基对的延伸的反密码子茎的tRNA前体容易被小麦和烟草剪接核酸内切酶接受为底物，而可以形成仅两个推定碱基对的延伸的反密码子茎的前tRNA分子根本不被剪接。琥珀抑制，从含内含子的延伸tRNA（Met）基因产生的，是有效地处理和剪接在两种植物提取物。
Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA(G PsiA)(Tyr) and elongator tRNA(M)et(C)mAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA(Tyr). Here we have studied the expression of an Arabidopsis elongator tRNA(Met) gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA(Met) precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA(Met) to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3' and 5' splice sites and of a structured intron for pre-tRNA(Met) splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA(Met) splicing and that a highly structured intron is indispensable for pre-tRNA(Met) splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA(Met) gene, is efficiently processed and spliced in both plant extracts.