A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro

Cell. 1996 Mar 8;84(5):801-11. doi: 10.1016/s0092-8674(00)81057-0.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Composition
  • Base Sequence
  • Blotting, Northern
  • Cell Nucleus / metabolism
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides
  • Plasmids
  • Polymerase Chain Reaction
  • RNA Precursors / isolation & purification
  • RNA Precursors / metabolism*
  • RNA Splicing*
  • Ribonuclease H
  • Ribonucleoprotein, U5 Small Nuclear / isolation & purification
  • Ribonucleoprotein, U5 Small Nuclear / metabolism*
  • Ribonucleoproteins, Small Nuclear / isolation & purification
  • Ribonucleoproteins, Small Nuclear / metabolism*

Substances

  • Oligodeoxyribonucleotides
  • RNA Precursors
  • Ribonucleoprotein, U5 Small Nuclear
  • Ribonucleoproteins, Small Nuclear
  • U11 small nuclear ribonucleoprotein, human
  • Ribonuclease H