IDENTIFICATION AND FUNCTIONAL-ANALYSIS OF 2 U3 BINDING-SITES ON YEAST PRERIBOSOMAL RNA
IDENTIFICATION AND FUNCTIONAL-ANALYSIS OF 2 U3 BINDING-SITES ON YEAST PRERIBOSOMAL RNA
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DOI：
10.1002/j.1460-2075.1992.tb05198.x
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发表时间：
1992-04-01
期刊：
影响因子：
11.4
通讯作者：
TOLLERVEY, D
中科院分区：
文献类型：
--
作者：
BELTRAME, M;TOLLERVEY, D
人们早就知道 U3 可以与前核糖体 RNA 形成氢键，但相互作用位点的特征却很少。在这里，我们证明酵母 U3 可以在脱蛋白提取物和活细胞中与 35S pre-rRNA 交联。交联位点定位于 5' 外部转录间隔区 (ETS)，然后在核苷酸水平上进行鉴定。 U3 靠近 5' 端的两个区域在体内和体外与 pre-rRNA 交联；进化上保守的 Box A 区域和与 ETS 序列完全互补的 10 个核苷酸 (nt) 序列。在 ETS 中检测到两个体内交联，位于 +470 处（与 U3 互补的区域内）和位于 +655 处，靠近 18S rRNA 5' 端的切割位点。带标签的 rDNA 构建体用于追踪体内 ETS 突变的影响。 ETS 中 +470 交联位点周围的小缺失会阻止 18S rRNA 的合成。该区域与脊椎动物 ETS 裂解位点同源。我们认为该位点可能在进化上是保守的，以指导产生 18S rRNA 的裂解所需的前 rRNA 加工复合物的组装。
It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.