NUCLEOTIDE-SEQUENCE DETERMINING THE 1ST CLEAVAGE SITE IN THE PROCESSING OF MOUSE PRECURSOR RIBOSOMAL-RNA
NUCLEOTIDE-SEQUENCE DETERMINING THE 1ST CLEAVAGE SITE IN THE PROCESSING OF MOUSE PRECURSOR RIBOSOMAL-RNA
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DOI：
10.1073/pnas.84.3.629
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发表时间：
1987-02-01
影响因子：
11.1
通讯作者：
SOLLNERWEBB, B
中科院分区：
文献类型：
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作者：
CRAIG, N;KASS, S;SOLLNERWEBB, B
小鼠细胞中47S前体rRNA加工的第一步是在体外S-100转录反应中复制，包括初级转录物残基+650处的核内裂解，然后是残基+650上游片段的快速降解。类似的过程也发生在人类rRNA中。小鼠和人的rRNA序列为。apprxeq。在这些加工位点的3'侧，200个核苷酸保守80%，这表明这个保守区域可能在指定加工过程中很重要。为了验证这一假设，我们构建了一系列系统的从5‘’和3‘’方向接近小鼠rDNA加工区域的缺失突变体，并在体外分析了其转录本的加工。加工所需区域的5”边界相当尖锐，与保守序列区域5”末端的rRNA切割位点相对应。3 “边界更为复杂：(i)加工位点以外延伸至250至130个核苷酸的3 ”缺失导致加工RNA的数量减少约50%。（ii）在加工位点之外延伸到109个核苷酸的3‘’缺失大大降低了加工效率。（iii）加工位点3'侧的91个或以上核苷酸的缺失实际上消除了加工。在改变的离子条件下，3‘’缺失的转录本仅延伸到加工位点以外的41个核苷酸，仍然可以指导低水平的精确加工。这些结果表明，小鼠/人类在主要rRNA加工位点3'侧的保守序列由几个结构域组成，这些结构域指导和/或增强了初始的核内裂解和上游片段的紧密耦合选择性降解，共同构成主要rRNA加工事件。
The first step in the processing of 47S precursor rRNA in mouse cells is reproduced in vitro in an S-100 transcription reaction and consists of an endonucleolytic cleavage at residue +650 of the primary transcript followed by rapid degradation of the fragment upstream from residue +650. An analogous processing occurs in human rRNA. The mouse and human rRNA sequences are .apprxeq. 80% conserved for 200 nucleotides on the 3'' side of these processing sites, suggesting that this conserved region may be important in specifying the processing. To test this hypothesis, we constructed a systematic series of deletion mutants approaching the mouse rDNA processing region from both the 5'' and 3'' directions and analyzed the processing of their transcripts in vitro. The 5'' boundary of the region required for processing is quite sharp and corresponds to the rRNA cleavage site at the 5'' end of the conserved sequence region. The 3'' boundary is more complex: (i) The 3'' deletions extending to between 250 and 130 nucleotides beyond the processing site cause about a 50% decrease in the amount of the processed RNA. (ii) A 3'' deletion that extends to 109 nucleotides beyond the processing site greatly reduces the processing efficiency. (iii) Deletions to or beyond 91 nucleotides on the 3'' side of the processing site virtually eliminate processing. Under altered ionic conditions, transcripts of 3'' deletions extending to only 41 nucleotides beyond the processing site can still direct a low level of accurate processing. These results demonstrate that the mouse/human conserved sequence just on the 3'' side of the primary rRNA processing site consists of several domains that direct and/or augment both the initial endonucleolytic cleavage and the closely coupled selective degradation of the upstream fragment that together constitute the primary rRNA processing event.