| Abstrakt: |
U1 small nuclear ribonucleoprotein (snRNP) may function during several steps of spliceosome assembly. Most spliceosome assembly assays, however, fail to detect the U1 snRNP. Here, I used a new native gel electrophoretic assay to find the yeast U1 snRNP in three pre-splicing complexes (δ, β1, α2) formed in vitro. The order of complex formation is deduced to be δ → β1→ α2→ α1→ β2,the active spliceosome. The δ complex is formed when U1 snRNP binds to pre-mRNA in the absence of ATP. There are two forms of δ: a major one, δun,unstable to competitor RNA; and a minor one, δcommit, committed to the splicing pathway. The other complexes are formed in the presence of ATP and contain the following snRNPs: β1, the pre-spliceosome, has both U1 and U2; α2has all five, however, U1 is reduced compared with the others; and α1and β2have U2, U5, and U6. Prior work by others suggests that U1 is “handing off” the 5′ splice site region to the U5 and U6 snRNPs before splicing begins. The reduced levels of U1 snRNP in the α2complex suggests that the handoff occurs during formation of this complex. |
