MAPPING THE 5' AND 3' ENDS OF TETRAHYMENA-THERMOPHILA MESSENGER-RNAS USING RNA LIGASE MEDIATED AMPLIFICATION OF CDNA ENDS (RLM-RACE)
MAPPING THE 5' AND 3' ENDS OF TETRAHYMENA-THERMOPHILA MESSENGER-RNAS USING RNA LIGASE MEDIATED AMPLIFICATION OF CDNA ENDS (RLM-RACE)
复制标题
DOI：
10.1093/nar/21.21.4954
复制
发表时间：
1993-10-25
影响因子：
14.9
通讯作者：
GOROVSKY, MA
中科院分区：
文献类型：
--
作者：
LIU, XW;GOROVSKY, MA
描述了用于绘制RNA末端的程序。使用T4 RNA连接酶，将DNA（3'末端）或RNA（5'末端）寡核苷酸连接到RNA末端，然后进行cDNA合成、PCR扩增、克隆和测序。该方法确定了5'末端、3'多聚腺苷酸化位点和poly（A）尾的大小，并且应该适用于非多聚腺苷酸化的mRNA和非信使RNA。四种嗜热四膜虫组蛋白mRNA的分析揭示了多个紧密间隔的5'末端，与通过其他方法确定的那些一致。除了在转录起始位点上游100-200个核苷酸的任一方向上的“CCAAT”盒外，在非翻译5'区或紧邻转录起始位点侧翼的序列中未观察到保守序列元件。对编码四种组蛋白、两种微管蛋白和四膜虫TATA结合蛋白的mRNA的3'末端的分析证实了四膜虫组蛋白信息是多腺苷酸化的并且该生物体中的poly（A）尾是短的（约50 nt）的观察结果。没有发现典型的poly（A）加成信号，这里分析的四个组蛋白信息包含三个序列元件，TGTGT-TAAAAGTATT，在非组蛋白信息中没有发现。两个非组蛋白信息在poly（A）添加位点附近含有GCATT（N）15 ATACC。
A procedure is described for mapping the ends of RNAs. Using T4 RNA ligase, a DNA (3' end) or RNA (5' end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing. This method determines 5' ends, 3' polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs. Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5' ends consistent with those determined by other methods. Except for a 'CCAAT' box in either orientation 100-200 nucleotides upstream of the transcription start site, no conserved sequence elements were observed in the untranslated 5' region or in sequences immediately flanking the transcription start site. Analysis of the 3' ends of mRNAs encoding four histones, two tubulins and the Tetrahymena TATA binding protein confirmed the observations that Tetrahymena histone messages are polyadenylated and that poly(A) tails in this organism are short (approximately 50 nt). No canonical poly(A) addition signal was identified.The four histone messages analyzed here contained three sequence elements, TGTGT-TAAAAGTATT, not found in non-histone messages. Two non-histone messages contained GCATT(N)15ATACC near the poly(A) addition site.