Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3′ ETS but not the 5′ ETS
Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3′ ETS but not the 5′ ETS
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DOI：
10.1017/s135583829999026x
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发表时间：
1999-07-01
期刊：
影响因子：
4.5
通讯作者：
Tollervey, D
中科院分区：
文献类型：
--
作者：
Kufel, J;Dichtl, B;Tollervey, D
我们重新审视了酵母 RNase III（Rnt1p）在核糖体合成中的作用。对携带 RNT1 基因完全缺失的菌株中前 rRNA 加工的分析表明，Rnt1p 的缺失不会阻止 5' 外部转录间隔区 (ETS) 中位点 A(0) 的切割，尽管位点 A(0)、A(1) 和 A(2) 处的早期前 rRNA 切割在动力学上延迟。相反，在没有 Rnt1p 的情况下，3' ETS 中的切割被完全抑制，导致 25S rRNA 3' 延伸形式的合成水平降低。 pre-rRNA 的 3' 延伸形式与位点 T2 (+210) 的主要终止一致。我们得出结论，Rnt1p 是 3' ETS 切割所必需的，但 Ao 位点切割不需要。通过引物延伸绘制了 3' ETS 体内切割位点的图谱。鉴定出两个 Rnt1p 依赖性裂解位点，位于预测的茎环结构的相对侧，即 +14 和 +49。这些与共有的 Rnt1p 切割位点非常一致。发现野生型细胞中成熟 25S rRNA 序列 3' 端的加工与 5.8S rRNA 5' 端的加工同时发生，这支持了之前关于 ITS1 和 3' ETS 中的加工是耦合的提议。
We have reexamined the role of yeast RNase III(Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A(0) in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A(0), A(1), and A(2) are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site Ao. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.