SPF30 is an essential human splicing factor required for assembly of the U4/U5/U6 tri-small nuclear ribonucleoprotein into the spliceosome.
Spliceosome assembly involves the sequential recruitment of small nuclear ribonucleoproteins (snRNPs) onto a pre-mRNA substrate. Although several non-snRNP proteins function during the binding of U1 and U2 snRNPs, little is known about the subsequent binding of the U4/U5/U6 tri-snRNP. A recent proteomic analysis of the human spliceosome identified SPF30 (Neubauer, G., King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P., Sleeman, J., Lamond, A., and Mann, M. (1998) Nat. Genet. 20, 46-50), a homolog of the survival of motor neurons (SMN) protein, as a spliceosome factor. We show here that SPF30 is a nuclear protein that associates with both U4/U5/U6 and U2 snRNP components. In the absence of SPF30, the preformed tri-snRNP fails to assemble into the spliceosome. Mass spectrometric analysis shows that a recombinant glutathione S-transferase-SPF30 fusion protein associates with complexes containing core Sm and U4/U5/U6 tri-snRNP proteins when added to HeLa nuclear extract, most strongly to U4/U6-90. The data indicate that SPF30 is an essential human splicing factor that may act to dock the U4/U5/U6 tri-snRNP to the A complex during spliceosome assembly or, alternatively, may act as a late assembly factor in both the tri-snRNP and the A-complex.
Rappsilber
J
J
Protein Interaction Laboratory, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Ajuh
P
P
Lamond
A I
AI
Mann
M
M
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000737:chemistry; D017362:Cyclic AMP Response Element-Binding Protein; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000502:physiology*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry*; D055532:SMN Complex Proteins; D017381:Spliceosomes / Q000737:chemistry*
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D055532:SMN Complex Proteins; C526478:SMNDC1 protein, human