SMNrp is an essential pre-mRNA splicing factor required for the formation of the mature spliceosome.
SMNrp, also termed SPF30, has recently been identified in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP-associated protein that appears in the pre-spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the first step of pre-mRNA splicing by preventing the formation of complex B. Re-addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6.U5] tri-snRNP. A candidate that may mediate this interaction, namely the U4/U6-90 kDa protein, has been identified. We suggest that SMNrp, as a U2 snRNP-associated protein, facilitates the recruitment of the [U4/U6.U5] tri-snRNP to the pre-spliceosome.
Meister
G
G
Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, University of Göttingen, Biochemie II, Herrmann Düker Weg 12, D-37073 Göttingen, Germany.
Hannus
S
S
Plöttner
O
O
Baars
T
T
Hartmann
E
E
Fakan
S
S
Laggerbauer
B
B
Fischer
U
U
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein; D006801:Humans; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D055532:SMN Complex Proteins; D017381:Spliceosomes / Q000378:metabolism*; D014158:Transcription, Genetic; D014981:Xenopus
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; C526478:SMNDC1 protein, human; C105252:tri-snRNP-specific protein, 27-kDa, human