SEQUENCE AND STRUCTURAL ELEMENTS CRITICAL FOR U8 SNRNP FUNCTION IN XENOPUS OOCYTES ARE EVOLUTIONARILY CONSERVED
SEQUENCE AND STRUCTURAL ELEMENTS CRITICAL FOR U8 SNRNP FUNCTION IN XENOPUS OOCYTES ARE EVOLUTIONARILY CONSERVED
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DOI：
10.1101/gad.8.18.2241
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发表时间：
1994-09-15
影响因子：
10.5
通讯作者：
STEITZ, JA
中科院分区：
文献类型：
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作者：
PECULIS, BA;STEITZ, JA
我们在非洲爪蟾 U8 RNA（5.8S 和 28S rRNA 成熟所需的核仁 snRNA）中生成了突变体，以鉴定对 RNA 稳定性、颗粒组装和 U8 RNP 功能至关重要的序列和结构域。通过将体外合成的 U8 RNA 显微注射到非洲爪蟾卵母细胞的细胞质中来测定突变体的活性。大多数突变 RNA 是稳定的，结合纤维蛋白（一种几种核仁特异性 snRNP 共有的蛋白质），并且变得高度甲基化。尽管 U8 RNA 5' 帽的超甲基化和纤维蛋白结合可能发生在非洲爪蟾卵母细胞的细胞质或核区室中，但这两者都不是核输入所必需的。我们发现，三甲基鸟苷帽虽然存在于内源性 U8 RNA 上，但对于 U8 在 28S RNA 的 3' 位点和 5.8S RNA 的 5' 位点的前 rRNA 协调加工中的稳定性、颗粒组装或功能来说并不是必需的。 U8 RNA 5' 结构域内的几个保守的单链和双链序列对于功能至关重要。
We have generated mutants in Xenopus U8 RNA, a nucleolar snRNA required for the maturation of 5.8S and 28S rRNAS, to identify sequences and structural domains essential for RNA stability, particle assembly, and function of the U8 RNP. Activity of the mutants was assayed by microinjection of in vitro-synthesized U8 RNAs into the cytoplasm of Xenopus oocytes. Most of the mutant RNAs were stable, bound fibrillarin, a protein common to several of the nucleolar-specific snRNPs, and became hypermethylated. Although hypermethylation of the 5' cap of U8 RNA and fibrillarin binding can occur in either the cytoplasmic or nuclear compartment of Xenopus oocytes, neither is required for nuclear import. We find that the trimethylguanosine cap, although present on the endogenous U8 RNA, is not essential for stability, particle assembly, or functioning of U8 in the coordinate processing of pre-rRNA at sites 3' of 28S and 5' of 5.8S RNA. Several conserved single- and double-stranded sequences within the 5' domain of U8 RNA are essential for function.