Peculis B A, Steitz J A
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University Medical School, New Haven, CT 06536.
Genes Dev. 1994 Sep 15;8(18):2241-55. doi: 10.1101/gad.8.18.2241.
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.
我们已在非洲爪蟾U8 RNA（一种5.8S和28S rRNA成熟所需的核仁小核仁RNA）中产生突变体，以鉴定对U8核糖核蛋白颗粒（RNP）的RNA稳定性、颗粒组装及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检测突变体的活性。大多数突变RNA是稳定的，能结合纤维蛋白原（一种几种核仁特异性小核仁核糖核蛋白共有的蛋白质），并发生超甲基化。尽管U8 RNA的5'帽超甲基化和纤维蛋白原结合可发生在非洲爪蟾卵母细胞的细胞质或细胞核区室中，但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的3'和5.8S RNA的5'位点对前体rRNA进行协同加工时的稳定性、颗粒组装或功能并非必不可少。U8 RNA 5'结构域内的几个保守单链和双链序列对其功能至关重要。
