Inhibitory effects of expanded GAA•TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo
Inhibitory effects of expanded GAA•TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo
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DOI：
10.1074/jbc.273.23.14588
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发表时间：
1998-06-05
影响因子：
4.8
通讯作者：
Pandolfo, M
中科院分区：
文献类型：
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作者：
Ohshima, K;Montermini, L;Pandolfo, M
弗里德赖希共济失调（FRDA）与共济失调蛋白基因第一内含子中GAA.TTC三联体重复序列的扩增有关，导致共济失调蛋白mRNA和蛋白水平降低。为了研究内含子扩展产生其效应的机制，在报告基因的内含子中以两个方向克隆了不同长度的GAA.TTC重复序列（9至270个三联体）。将含有这些重复序列的质粒瞬时转染COS-7细胞。观察到报告基因表达的长度和方向依赖性抑制。RNA酶保护和北方印迹分析表明，当较长的GAA重复序列被转录时，成熟mRNA的水平非常低，初级转录物没有积累。与携带（GAA.TTC）（9）和（GAA.TTC）（90）的质粒相比，携带长GAA.TTC片段（类似于250个三联体）的质粒在COS-7细胞中的复制受到极大抑制。转录物含有（GAA）的质粒（230）的复制抑制作用是转录物含有（UUC）的质粒（270）的5倍。我们的体内研究表明，扩大GAA。来自FRDA基因内含子I的TTC重复序列抑制转录而不是转录后RNA加工，并且也干扰复制。这些效应的分子基础可能是非B DNA结构的形成。
Friedreich ataxia (FRDA) is associated with the expansion of a GAA.TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA.TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA.TTC tracts (similar to 250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA.TTC)(9) and (GAA.TTC)(90). Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)(230) than for the plasmid whose transcript contains (UUC)(270). Our in vivo investigation revealed that expanded GAA . TTC repeats from intron I of the FRDA gene inhibit transcription rather than posttranscriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures.