The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant
The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant
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DOI：
10.1242/jcs.03288
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发表时间：
2006-12
影响因子：
4
通讯作者：
K. Sugaya;E. Hongo;Y. Ishihara;H. Tsuji
中科院分区：
文献类型：
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作者：
K. Sugaya;E. Hongo;Y. Ishihara;H. Tsuji
温度敏感型CHO-K1突变细胞系TsTM18表现出染色体不稳定和细胞周期停滞在S和G2期，在39℃的不允许温度下DNA合成减少。我们先前在Smu1中发现了一个氨基酸替代，这是TsTM18细胞温度敏感表型的基础。在本研究中，我们通过RNA干扰证实了Smu1与tsTM18的温敏缺陷有关。我们还发现了DNA合成的早期温度效应。由于线虫的遗传学研究表明SMU-1参与了unc52/perlecan前mRNA的剪接，因此我们利用逆转录-聚合酶链式反应(RT-PCR)分析了tsTM18细胞中的perlecan转录本。从39℃培养的tsTM18细胞的RNA中扩增出的Perlecan PCR产物似乎是一种变异体的混合物。序列分析确定了至少6个变异体，它们是由选择性剪接和内含子保留引起的。将Perlecan RT-PCR分析结果与其他4个基因的分析结果进行比较，表明Perlecan基因的剪接缺陷是独一无二的，并且在进化过程中是保守的。
Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell-cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. We previously identified an amino acid substitution in Smu1 that underlies the temperature-sensitive phenotypes of tsTM18 cells. In the present study, we confirmed that Smu1 is associated with the temperature-sensitive defect of tsTM18 by RNA interference. We also found an early temperature effect in DNA synthesis. Because genetic studies of nematodes revealed that smu-1 is involved in splicing of the unc52/perlecan pre-mRNA, we analysed the perlecan transcript in tsTM18 cells by reverse transcription-polymerase chain reaction (RT-PCR). The perlecan PCR product amplified from RNA of tsTM18 cells cultured at 39°C appeared to be a mixture of variants. Sequence analysis identified at least six variants that result from alternative splicing and intron retention. Comparison of the results of perlecan RT-PCR analysis with those of analysis of four other genes suggested that the splicing defect in the perlecan gene is unique and that it is conserved through evolution.