Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells
Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells
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DOI：
10.1073/pnas.231490998
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发表时间：
2001-11-06
影响因子：
11.1
通讯作者：
Steitz, JA
中科院分区：
文献类型：
--
作者：
Hirose, T;Steitz, JA
在哺乳动物细胞中，所有指导 rRNA 修饰的小核仁 RNA (snoRNA) 均在宿主基因的内含子内编码。对人类 Box C/D snoRNA 的数据库分析揭示了其内含子位置的保守性，优先选择 3' 剪接位点上游的 70-80 nt。转染实验表明，对于 snoRNA 和 3' 剪接位点之间具有更长或更短间隔区的突变体构建体，gas5 编码的 U75 和 U76 snoRNA 的合成显着下降。然而，snoRNA的位置并不影响宿主内含子的剪接。间隔区内的取代突变表明长度而不是特定序列很重要。一种将 U75 的前 mRNA 剪接和加工耦合的体外系统已经开发出来。 U75 体外合成取决于其盒 C 和 D 序列，并且需要适当的间隔区长度。进一步的体内和体外突变分析以及随后的分支点图谱表明，关键距离是从 snoRNA 编码区到分支点，这表明剪接和 snoRNA 释放之间存在协同作用。
In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. A database analysis of human box C/D snoRNAs revealed conservation of their intronic location, with a preference for 70-80 nt upstream of the 3' splice site. Transfection experiments showed that synthesis of gas5-encoded U75 and U76 snoRNAs dropped significantly for mutant constructs possessing longer or shorter spacers between the snoRNA and the 3' splice site. However, the position of the snoRNA did not affect splicing of the host intron. Substitution mutations within the spacer indicated that the length, but not the specific sequence, is important. A in vitro system that couples pre-mRNA splicing and processing of U75 has been developed. U75 synthesis in vitro depends on its box C and D sequences and requires an appropriate spacer length. Further mutational analyses both in vivo and in vitro, with subsequent mapping of the branch points, revealed that the critical distance is from the snoRNA coding region to the branch point, suggesting synergy between splicing and snoRNA release.