Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles.
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 of the U2/U6 cross-link with 254 nm irradiation released 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 oligonucleotide "splint" technique. The labeled U2/U6 complex was then subjected to rapid enzymatic RNA sequencing or to targeted digestion 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 upstream 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 brucei, 8 in Pisum sativum, 11 in Saccharomyces cerevisiae) is significant.
Hausner
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TP
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510-8024.
Giglio
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LM
Weiner
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AM
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D015152:Blotting, Northern; D003432:Cross-Linking Reagents; D004722:Endoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D010802:Phylogeny; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics; D016914:Ribonuclease H; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C018822:aminomethyltrioxsalen; D004722:Endoribonucleases; D016914:Ribonuclease H; D014307:Trioxsalen