An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2
An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2
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DOI：
10.1093/emboj/16.13.4092
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发表时间：
1997-07-01
期刊：
影响因子：
11.4
通讯作者：
Luhrmann, R
中科院分区：
文献类型：
--
作者：
Fabrizio, P;Laggerbauer, B;Luhrmann, R
剪接体中发生的许多RNA构象变化背后的驱动力尚不清楚。在这里，我们描述了一个进化上保守的人类U5小核糖核蛋白(SnRNP)蛋白(U5-116kD)，它与核糖体延长因子EF-2(核糖体转位酶)具有惊人的同源性。在酿酒酵母中鉴定出一个与U5-116kD同源的114 kDa蛋白(Snu114p)，它是酵母细胞生存所必需的。Snu114p的遗传缺失导致未剪接Pre-mRNA的积累，表明Snu114p是体内剪接所必需的。针对U5-116kD的抗体在体外抑制HeLa核提取液中的前mRNA剪接。在HeLa细胞中，U5-116kD位于细胞核内，并与含有SnRNP的亚核结构共存，称为斑点。U5-116kD/Snu114p的G结构域包含与GTP结合和水解重要的共同序列元件G1-G5。与此一致的是，U5-116kD可以通过U5 SnRNPs的紫外光照射而与GTP特异地交联。此外，Snu114p的G1序列基序中的单个氨基酸替换是致命的，有望取消GTP结合活性，这表明GTP结合以及可能的GTP水解对U5-116kD/Snu114p的功能是重要的。到目前为止，这是第一个证据表明包含G结构域的蛋白在前mRNA剪接过程中发挥重要作用。
The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.