Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.
The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.
Krämer
A
A
Département de Biologie Cellulaire, Université de Genève, CH-1211 Genève 4, Switzerland. angela.kraemer@cellbio.unige.ch
Grüter
P
P
Gröning
K
K
Kastner
B
B
D000837:Anion Exchange Resins; D001483:Base Sequence; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D008836:Micrococcal Nuclease; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012117:Resins, Synthetic; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure*; D017381:Spliceosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D000837:Anion Exchange Resins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012117:Resins, Synthetic; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human; C088049:splicing factor 3a; C042855:Mono Q; D008836:Micrococcal Nuclease