Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro.
Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro.
复制标题
酵母 U6 snRNA 中的位点特异性脱氧核苷酸取代可在体外阻断前 mRNA 的剪接。
DOI：
10.1093/emboj/16.8.2119
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Abelson,J
中科院分区：
文献类型：
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作者：
Kim,CH;Ryan,DE;Marciniec,T;Abelson,J
我们已经确定了U6磷酸核糖骨架的2 ′-羟基，这是在U6缺失的酵母提取物中重建剪接活性所必需的。为了筛选酵母U6在核苷酸39 - 88处的2 ′-羟基，跨越保守的中心结构域，合成U6 RNA与脱氧核糖核苷酸特异性地掺入。只有四个单独的脱氧核苷酸取代阻断了剪接活性：dA51（在ACAG A G序列中），dA62（在AGC三联体旁边），dU70和dC72（都在3 ′分子内茎环的环中）。天然凝胶分析显示，这些脱氧取代的U6 RNA能够组装剪接体。有趣的是，A51、A62、U70或C72上的2 ′-O-甲基取代基并不抑制剪接活性，这表明U6中这些位置上的必需2 ′-OH基团充当氢键受体或中性配位配体。在A62、U70和C72处的必需2 ′-羟基相对于II组核酶的催化结构域V的必需2 ′-羟基的位置显示出相似性和差异性。在位置62、70和72处的必需2 ′-羟基的鉴定证实了U6中的3 ′分子内茎环在前mRNA剪接中起着重要作用。
We have identified 2′‐hydroxyl groups of the U6 phosphate‐ribose backbone which are required for reconstitution of splicing activity in U6‐depleted yeast extract. To screen the 2′‐hydroxyls of yeast U6 at nucleotides 39–88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically. Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAG A G sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3′ intramolecular stem–loop). Native gel analysis revealed that these deoxy‐substituted U6 RNAs were competent for assembly of spliceosomes. Interestingly, a 2′‐O‐methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2′‐OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands. The requisite 2′‐hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2′‐hydroxyls of catalytic domain V of group II ribozymes. The identification of the essential 2′‐hydroxyls at positions 62, 70 and 72 corroborates that the 3′ intramolecular stem–loop in U6 plays an important role in pre‐mRNA splicing.