PATHWAY OF CYTOCHROME-B MESSENGER-RNA PROCESSING IN YEAST MITOCHONDRIA - SPECIFIC SPLICING STEPS AND AN INTRON-DERIVED CIRCULAR RNA
PATHWAY OF CYTOCHROME-B MESSENGER-RNA PROCESSING IN YEAST MITOCHONDRIA - SPECIFIC SPLICING STEPS AND AN INTRON-DERIVED CIRCULAR RNA
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DOI：
10.1016/0092-8674(80)90506-1
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发表时间：
1980-01-01
期刊：
影响因子：
64.5
通讯作者：
SLONIMSKI, P
中科院分区：
文献类型：
--
作者：
HALBREICH, A;PAJOT, P;SLONIMSKI, P
利用基因工程技术研究了编码细胞色素B的嵌合线粒体基因cob-box在S.酿酒厂进行了研究。采用Alwine、肯普和Stark（1977）的技术，使用含有部分细胞色素B基因的DNA片段和EM分析线粒体RNA。只有高分子量，不完全加工的转录本中观察到的内含子突变体时，使用羧基末端外显子探针和氨基近端内含子探针，而外显子突变体含有2.7 kb（内切酶对）的mRNA不杂交的内含子探针。8.6、8.1和7.1 kb的物质分别在box 8、box 3和box 10突变体中积累。前2个物种的加工也分别以0.8和0.9 kb稳定内含子RNA（1 kb RNA）的顺序出现为标志。因此，通过内含子突变中断mRNA加工，可以推导出细胞色素B mRNA成熟的途径，其中至少一些内含子以超过1步的顺序剪接。在3个rho- petites中也观察到1 kb RNA，其补充了box 3突变。在2个非互补的小片段中没有发现它们，尽管这些小片段也含有野生型box 3等位基因并产生mtDNA转录本。1 kb RNA图谱位于从box 4延伸到box 1的氨基近端内含子内，但位于包含box 3突变的片段外。1 kb RNA部分由环状RNA分子0.275 ± 0.275组成。0.013 μ m长和线性分子0.238 ±。0.028 μ m长。在这些小叶柄中也观察到较大的转录本，表明小叶柄能够进行转录本加工。内含子中的正常核苷酸序列及其侧翼位点对于正确剪接的发生是必要的，但不充分。其他与之相距甚远的序列也很重要。剪接的发生和发展很可能涉及一个特定的三维结构，可以识别的酶机制的时间序列。使这些结构变形的突变和通过RNA互补的校正可能是更好地理解这种现象的有力工具。
The expression of the mosaic mitochondrial gene cob-box, which codes for cytochrome b in S. cerevisiae was studied. Mitochondrial RNA were analyzed by the technique of Alwine, Kemp and Stark (1977) using DNA fragments containing parts of the cytochrome b gene and by EM. Only high MW, not fully processed transcripts are observed in intron mutants when a carboxy terminal exon probe and an amino proximal intron probe are used, whereas exon mutants contain 2.7 kb (kilobase-pair) mRNA which does not hybridize to the intron probe. The 8.6, 8.1 and 7.1 kb species accumulate in box8, box3 and box10 mutants, respectively. The processing of the first 2 species is also marked by the sequential appearance of 0.8 and 0.9 kb stable intron RNA, respectively (1 kb RNA). The interruption of mRNA processing by intron mutations thus allowed the deduction of a pathway for the maturation of cytochrome b mRNA in which introns are sequentially spliced with more than 1 step for at least some of them. The 1 kb RNA were also observed in 3 rho- petites which complement box3 mutations. They were not found in 2 noncomplementing petites even though these also contain the wild-type box3 allele and produce mtRNA transcripts. The 1 kb RNA map inside the amino proximal intron that extends from box4 to box1, but outside the segment encompassing box3 mutations. The 1 kb RNA fraction consists of circular RNA molecules 0.275 .+-. 0.013 .mu.m long and linear molecules 0.238 .+-. 0.028 .mu.m long. Larger transcripts were also observed in these petites, indicating that petites are capable of transcript processing. The normal sequence of nucleotides in an intron and its flanking sites is necessary but insufficient for the correct splice to occur. Other sequences quite remote from it are also important. The onset and progression of splicing most probably involves a temporal succession of specific 3-dimensional structures that can be recognized by the enzymic machinery. Mutations that deform these structures and corrections by RNA complementation may be a powerful tool for a better understanding of the phenomenon.