Liang W Q, Clark J A, Fournier M J
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003, USA.
Mol Cell Biol. 1997 Jul;17(7):4124-32. doi: 10.1128/MCB.17.7.4124.
The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.
系统发育保守的U14小核仁RNA是rRNA加工所必需的，该功能涉及与18S RNA中保守的互补序列进行碱基配对。为了鉴定其他重要的U14相互作用，首先对酿酒酵母U14 RNA活性所需的茎环结构域（Y结构域）进行了详细的突变分析。定位结果表明，Y结构域的大多数核苷酸可以被替换而不影响功能，除了在五个不同酵母物种中保守的环核苷酸。然后使用缺陷变体来鉴定基因内和基因外抑制突变。所有基因内突变都定位在原发突变的六个核苷酸范围内，这表明抑制涉及构象变化，并且环元件参与了关键的分子间相互作用，而不是分子内碱基配对。一个高拷贝的基因外抑制基因，命名为DBP4（DEAD盒蛋白4），编码DEAD-DEXH盒家族的一个必需的推定RNA解旋酶。DBP4的抑制作用（最初称为CA4 [张.-H.，阿雷纳斯，J.和阿贝尔森，J.，美国国家科学院院刊87:1571-1575，1990]）恢复了18S rRNA的水平，对Y结构域具有特异性，但不具有等位基因特异性。预计DBP4要么在U14小核仁核糖核蛋白的组装中起作用，要么更有可能在其与rRNA加工装置的其他组分的相互作用中起作用。介导U14与前体18S RNA的相互作用是一种特别有吸引力的可能性。
