Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease
Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease
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DOI：
10.1093/emboj/20.23.6856
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发表时间：
2001-12-03
期刊：
影响因子：
11.4
通讯作者：
Bozzoni, I
中科院分区：
文献类型：
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作者：
Giorgi, C;Fatica, A;Bozzoni, I
一个外部茎，对于小核仁rna （snoRNAs）从它们的前mrna中释放是必不可少的，位于大多数酵母内含子编码的snoRNAs的两侧。即使该茎不是典型的Rnt1p底物，一些实验表明Rnt1p内切酶是snoRNA加工所必需的。为了确定处理内含子编码的snoRNAs所需的因素，我们在体外培养了能够重现这种活性的提取物。我们发现snoRNP因子在所有加工步骤中都与snorna编码区相关，并且无法组装snoRNP的突变体具有加工缺陷表型。Nop1p的特异性缺失完全阻止了U18 snoRNA的合成，但不影响具有典型Rnt1p位点的双双电子snoRNA编码单元的加工。通过将纯化的Nop1p和Rnt1p一起孵育，可以在体外复制内含子编码的U18和snR38 snoRNAs的正确切割。下拉实验表明，这两种蛋白质在物理上相互作用。这些数据表明，U18、snR38以及可能的其他内含子编码的snoRNAs的切割是一个受调控的过程，因为只有当snoRNP组装发生时，茎才会被Rnt1p内切酶切割。
An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNAs. Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing. To identify the factors necessary for processing of intron-encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA-coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing-deficient phenotype. Specific depletion of Nop1p completely prevents U18 snoRNA synthesis but does not affect processing of a dicistronic snoRNA-coding unit that has a canonical Rnt1p site. Correct cleavage of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p. Pull-down experiments showed that the two proteins interact physically. These data indicate that cleavage of U18, snR38 and possibly other intron-encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred.