Dual nature of pseudouridylation in U2 snRNA: Pus1p-dependent and Pus1p-independent activities in yeasts and higher eukaryotes.
Dual nature of pseudouridylation in U2 snRNA: Pus1p-dependent and Pus1p-independent activities in yeasts and higher eukaryotes.
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DOI：
10.1261/rna.061226.117
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发表时间：
2017-07
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影响因子：
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通讯作者：
Gall JG
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作者：
Deryusheva S;Gall JG
脊椎动物U2 snRNA中43位的假尿苷是剪接体snRNA中最保守的转录后修饰之一;在不同门（包括真菌、昆虫和蠕虫）的U2 snRNA中，相同位置被假尿苷化。假尿苷合酶Pus 1 p单独作用于U2 snRNA，在酵母酿酒酵母和小鼠中形成这种假尿苷。此外，在S.酿酒酵母，Pus 1 p是该位置的唯一假尿苷合酶。使用体内酵母细胞系统，我们测试了Pus 1 p的酶活性，从裂殖酵母裂殖酵母，蠕虫秀丽隐杆线虫，果蝇黑腹果蝇，和青蛙热带爪蟾。我们证明了Pus 1 p来自C.当在酵母细胞中表达时，线虫对U2 snRNA没有酶活性，而在类似的实验中，酵母U2 snRNA的44位（相当于脊椎动物中的43位）是来自S.酿酒酵母，S.粟酒属、果蝇属、非洲爪蟾属和小鼠。然而，当我们分析来自Pus 1基因敲除小鼠的U2 snRNA和pus 1 Δ S.粟酒裂殖酵母菌株，我们不能检测到任何变化，其修饰模式相比，野生型U2 snRNAs。In S. pombe中，我们发现了一个新的盒H/ACA RNA编码下游的RPC 10基因，并通过实验验证了其指导RNA活性定位的U2 snRNA中的p43和p44。在脊椎动物中，我们发现SCARNA 8（也称为U92 scaRNA）除了先前确定的靶点U2-B134/B144之外，还可以指导U2-B143。
The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved post-transcriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast Saccharomyces cerevisiae and mouse. Furthermore, in S. cerevisiae, Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system, we tested enzymatic activity of Pus1p from the fission yeast Schizosaccharomyces pombe, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the frog Xenopus tropicalis. We demonstrated that Pus1p from C. elegans has no enzymatic activity on U2 snRNA when expressed in yeast cells, whereas in similar experiments, position 44 in yeast U2 snRNA (equivalent to position 43 in vertebrates) is a genuine substrate for Pus1p from S. cerevisiae, S. pombe, Drosophila, Xenopus, and mouse. However, when we analyzed U2 snRNAs from Pus1 knockout mice and the pus1Δ S. pombe strain, we could not detect any changes in their modification patterns when compared to wild-type U2 snRNAs. In S. pombe, we found a novel box H/ACA RNA encoded downstream from the RPC10 gene and experimentally verified its guide RNA activity for positioning Ψ43 and Ψ44 in U2 snRNA. In vertebrates, we showed that SCARNA8 (also known as U92 scaRNA) is a guide for U2-Ψ43 in addition to its previously established targets U2-Ψ34/Ψ44.