A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs.
The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.
Meister
G
G
Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
Bühler
D
D
Pillai
R
R
Lottspeich
F
F
Fischer
U
U
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein; D006367:HeLa Cells; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; D017381:Spliceosomes / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism; D055517:snRNP Core Proteins
D001324:Autoantigens; D017362:Cyclic AMP Response Element-Binding Protein; C108381:GEMIN2 protein, human; D009419:Nerve Tissue Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; C526417:SNRPN protein, human; D055517:snRNP Core Proteins; D000255:Adenosine Triphosphate