EVIDENCE FOR BASE-PAIRING BETWEEN MAMMALIAN U2 AND U6 SMALL NUCLEAR RIBONUCLEOPROTEIN-PARTICLES
EVIDENCE FOR BASE-PAIRING BETWEEN MAMMALIAN U2 AND U6 SMALL NUCLEAR RIBONUCLEOPROTEIN-PARTICLES
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DOI：
10.1101/gad.4.12a.2146
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发表时间：
1990-12-01
影响因子：
10.5
通讯作者：
WEINER, AM
中科院分区：
文献类型：
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作者：
HAUSNER, TP;GIGLIO, LM;WEINER, AM
在补骨脂素衍生物AMT存在下，用365 nm光照射HeLa核抽提物，产生分子内和分子间的SnRNA交联物。去蛋白后，用凝胶电泳法分离交联型RNA，用Northern blotting鉴定为异常迁移物种。除了U4/U6的SnRNA交联物外，我们还检测到了一个分子间的U2/U6交联物，以及几个明显的分子内U1、U2和U5交联物。为了定位U2/U6交联链，利用一种新的寡核苷酸“夹板”技术选择性地标记了纯化的U2/U6复合体中U2的3‘’端。然后对标记的U2/U6复合体进行快速的酶RNA测序，或通过RNaseH和一组互补的寡核苷酸对复合体的U2和U6组分进行靶向切割。U2/U6交联链位于U2中15号核苷酸的上游和U6中85号核苷酸的下游，这表明这些区域(人、果蝇和秀丽线虫中连续6个碱基对，庞氏裂殖酵母和布鲁氏锥虫中7个，豌豆中8个，酿酒酵母中11个)之间的系统发育保守碱基配对是重要的。
Intramolecular and intermolecular snRNA cross-links were generated by irradiating HeLa nuclear extract with 365 nm light in the presence of the psoralen derivative AMT. After deproteinization, cross-linked RNAs were resolved by gel electrophoresis and identified as anomalously migrating species by Northern blotting. In addition to the U4/U6 snRNA cross-link, we detected an intermolecular U2/U6 cross-link, as well as several apparently intramolecular U1, U2 and U5 cross-links Photoreversal f the U2/U6 cross-link with 254 nm irradiation release stoichiometric amounts of U2 and U6 snRNA. To localize the U2/U6 cross-link, the 3'' end of U2 in the purified U2/U6 complex was labeled selectively using a novel olignucleotide "splint" technique. The labeled U2/U6 complex was then subjected to rapid enzymatic RNA sequencing or to targeted digetion of the U2 and U6 components of the complex by RNase H and a panel of complementary oligonucleotides. The U2/U6 cross-link is located upsteram of nucleotide 15 in U2 and downstream of nucleotide 85 in U6, suggesting that the phylogenetically conserved base-pairing between these regions (6 consecutive base pairs in human, Drosophila melanogaster, and Caenorhabditis elegans, 7 in Schizosaccharomyces pombe and Trypanasoma bruce, 8 in Pisum sativum, 11 in Saccharomyces cerevisiae) is significant.