The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex.
Recognition of the 5' splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5' splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5' splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5' splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5' splice site through base pairing with the 5' end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5' splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5' splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5' splice site in a mammalian preassembled penta-snRNP complex.
Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel.
Hadar
H
Malca
Noam
N
Shomron
Gil
G
Ast
D017398:Alternative Splicing; D001483:Base Sequence; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005363:Ficusin / Q000494:pharmacology; D005990:Glycerol / Q000378:metabolism / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D012333:RNA, Messenger; D017412:Ribonucleoprotein, U1 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D000511:alpha-Macroglobulins; D016914:Ribonuclease H; D008836:Micrococcal Nuclease; D005363:Ficusin; D005990:Glycerol