Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP
Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP
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DOI：
10.1128/mcb.22.23.8342-8352.2002
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发表时间：
2002-12-01
影响因子：
5.3
通讯作者：
Lührmann, R
中科院分区：
文献类型：
--
作者：
Watkins, NJ;Dickmanns, A;Lührmann, R
U4 snRNA的5'茎环和盒C/D snnas的盒C/D基序都可以折叠成一个类似的茎-内部环-茎结构，结合15.5K蛋白。同源蛋白NOP56、NOP58和61K （hPrp31）分别与盒型C/D snRNP和U4/U6 snRNP结合。这就提出了一个有趣的问题，即这两种同源RNP复合物是如何特异性地组装到类似的rna上的。在这里，我们研究了单个snoRNP蛋白在体外与U14盒C/D snoRNP特异性结合的要求。这表明，15.5K与盒C/D基序的结合对于剩余的snornp相关蛋白（即NOP56、NOP58、纤原蛋白以及核质蛋白TIP48和TIP49）的结合是必不可少的。盒状C/D基序的茎11，与U4 5'茎环相反，是高度保守的，我们发现该序列负责NOP56， NOP58，纤原蛋白，TIP48和TIP49与snoRNA结合，但不涉及15.5K。事实上，干细胞II序列对于微注射到HeLa细胞中的U14 snoRNA的核仁定位至关重要。因此，茎11的保守序列决定了box CID snoRNP的具体组装。
The 5' stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem 11 of the box C/D motif, in contrast to the U4 5' stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem 11 determines the specific assembly of the box CID snoRNP.