A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro.
A minor class of introns with noncanonical splice (AT-AC) and branch site sequences exists in metazoan protein coding genes. We have established a HeLa cell in vitro system that accurately splices a pre-mRNA substrate containing such an intron from the human P120 gene. Splicing occurs via a lariat intermediate whose branch site A residue is predicted to bulge from a duplex formed with the low abundance U12 small nuclear ribonucleoprotein (snRNP), which we confirm by psoralen cross-linking. Native gel electrophoresis reveals that U11, U12, and U5 snRNPs assemble onto the P120 pre-mRNA to form splicing complexes. Inhibition of P120 splicing by 2'-O-methyl oligonucleotides complementary to U12 or U5 demonstrates that U12 and U5 snRNPs perform essential roles in the AT-AC spliceosome.
Tarn
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WY
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Steitz
J A
JA
D001482:Base Composition; D001483:Base Sequence; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000302:isolation & purification / Q000378:metabolism*; D012326:RNA Splicing; D016914:Ribonuclease H; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000302:isolation & purification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C098762:U11 small nuclear ribonucleoprotein, human; D016914:Ribonuclease H