Small Cajal Body-specific RNAs of Drosophila Function in the Absence of Cajal Bodies
Small Cajal Body-specific RNAs of Drosophila Function in the Absence of Cajal Bodies
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DOI：
10.1091/mbc.e09-09-0777
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发表时间：
2009-12-15
影响因子：
3.3
通讯作者：
Gall, Joseph G.
中科院分区：
文献类型：
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作者：
Deryusheva, Svetlana;Gall, Joseph G.
在它们的生物发生过程中，小核RNA（snRNA）经历多个共价修饰，这需要引导RNA将甲基化酶和假尿苷酶引导至适当的核苷酸。由于它们位于核Cajal体（CB）中，这些指导RNA被称为小CB特异性RNA（scaRNA）。使用荧光引物延伸技术，我们绘制了果蝇U1，U2，U4和U5 snRNAs的修饰核苷酸。通过荧光原位杂交（FISH），我们发现，7果蝇scaRNA集中在容易检测的CB。我们使用了两种基于非洲爪蟾卵母细胞核的测定来证明这些果蝇scaRNA中的三种确实起着向导RNA的作用。在CB标记蛋白卷曲蛋白无效的果蝇中，不存在CB，并且不存在scaRNA的定位FISH信号。然而，生物化学实验表明scaRNA以正常水平存在，snRNA被适当修饰。我们的实验表明，几种scaRNA集中在野生型果蝇的CB中，但它们在缺乏CB的突变果蝇的核质中同样发挥作用。我们认为snRNA修饰机制并不局限于CB，而是分散在整个细胞的核质中。
During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.