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.Splicing of precursor mRNA (pre-mRNA) is a critical regulatory stage in which accurate recognition and removal of introns by the splicing machinery compose the correct code for protein production. The splicing reaction is carried out by the spliceosome, a dynamic complex of five small nuclear ribonucleoproteins (snRNPs)-U1, U2, U4, U5, and U6-and many auxiliary proteins. The spliceosome assembles de novo on each intron and, through a myriad of RNA-RNA, RNAprotein, and protein-protein interactions, acts to excise each intron and ligate the exons (reviewed in references 11, 20, 24, and 45).Spliceosome assembly requires specific recognition of splice site signals: the 5Ј splice site (5Јss) consensus sequence, which includes a conserved GU dinucleotide at the 5Ј end of the intron; the 3Ј splice site (3Јss) region, which consists of the polypyrimidine tract and a conserved AG dinucleotide at the 3Ј end of the intron; and the branch site located ϳ20 nucleotides upstream of the 3Јss, bearing a conserved adenosine (8).According to the current model of spliceosome assembly on pre-mRNA, the snRNPs join the spliceosome in an ordered pathway. U1 snRNP initially recognizes the 5Јss by base pairing between U1 snRNA and the 5Јss exon-intron junction (at positions Ϫ3 to ϩ6); these are highly complementary sequences (17,35,48,50,64). Non-snRNP splicing factors interact with the 3Јss, resulting in the 5Јss being brought to the proximity of the 3Јss. The U1/5Јss base pairing is then weakened in an ATP-dependent step (9,21,23,29,30,53), allowing U2 snRNP to base pair with the branch site. Next, the U4/U5/U6 trisnRNP complex is added, resulting in an apparent destabilization of U1 snRNP from the spliceosome (reviewed in reference 24), followed by several rearrangements in which U1 is replaced by U5 and U6 at the 5Јss (1,4,5,22,27,32). The U4/U6 base pairing within the U4/U5...

A multicomponent complex of proteins and RNA is assembled on the newly synthesized pre-mRNA to form the spliceosome. This complex catalyzes a two-step transesterification reaction required to remove the introns and ligate the exons. To date, only six proteins have been found necessary for the second step of splicing in yeast, and their human homologs have been identified. We demonstrate that the addition of the selective chelator of zinc, 1,10-phenanthroline, to an in vitro mRNA splicing reaction causes a dose-dependent inhibition of the second step of splicing. This inhibition is accompanied by the accumulation of spliceosomes paused before completion of step two of the splicing reaction. The inhibition effect on the second step is due neither to snRNA degradation nor to direct binding to the mRNA, and is reversible by dialysis or add-back of zinc, but not of other divalent metals, at the beginning of the reaction. These findings suggest that the activity of a putative zinc-dependent metalloprotein(s) involved in the second step of splicing is affected. This study outlines a new method for specific reversible inhibition of the second step of splicing using external reagents, and suggests a possible role of divalent cations in the second step of mRNA splicing, most likely zinc.