Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join Ul and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele ofprp2 (rna2). The isolated prp2A spliceosome contains U2, U5, U6, and possibly also I11 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.Splicing of introns from nuclear pre-mRNAs occurs by two cleavage-ligation (transesterification) reactions. The first reaction is a cleavage at the 5' splice site and the formation of a branched intron-exon 2 lariat molecule. The second reaction involves cleavage at the 3' splice site and ligation of the exons. For splicing to occur, the sequence-conserved regions in the intron (the branch site and the 5' and 3' splice sites) must be recognized and brought in close proximity. The interaction between splice sites is achieved through an ordered assembly of four small nuclear ribonucleoproteins (snRNPs) (Ul, U2, U5, and U4/U6) and protein factors on the pre-mRNA to form the spliceosome where splicing occurs (for reviews, see references 14, 21, 23, 28, 31, and 33).U4 and U6 RNAs reside in a single ribonucleoprotein particle (8, 16), and both are required for splicing in mammals and in yeast cells (3,5,9,32). During spliceosome assembly, Ul and U2 snRNPs bind to pre-mRNA at the early stages (27,30). The presplicing complex is converted to the spliceosome after the entry of U4/U6 and U5 snRNPs (12,17,24). Interestingly, prior to or concomitant with the 5' cleavage reaction, U4 RNA is no longer detected in the spliceosome when analyzed by native gel electrophoresis (12,17,24). However, it was suggested that U4 may not actually be released because a spliceosome containing spliced products can be affinity selected by using oligonucleotides complementary to U4 (6). It is possible that U4 is lost under the conditions of gel electrophoresis, which might indicate that the binding of U4 is destabilized during the assembly of the spliceosome. Nonetheless, it is still not clear whether U4 is involved only in the assembly of the spliceosome or whether it also has a catalytic function.One way to determine whether U4 RNA is present during the catalytic phase of splicing is to isolate a spliceosome prior to transesterification and to test whether U4 is both present and required for the splicing reaction. We took advantage of a yeast temperature-sensitive mutant, prp2, which is defective in pre-mRNA splicing (22, 26). Heatinactivated prp2 mutant extracts cannot catalyz...

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Studies of RNA-protein interactions often require assembly of the RNA-protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembled spliceosome is stable and can be isolated by sedimentation through glycerol gradients for subsequent analysis. The protocols describe two procedures to prepare RNA: using bacteriophage RNA polymerases or ligation of RNA oligos using T4 DNA ligase. We also describe the preparation of splicing competent yeast extracts, the assembly of the spliceosome, and the isolation of the spliceosome by glycerol gradient sedimentation. To allow exogenously added U6 RNA to be incorporated into the spliceosome, the endogenous U6 small nuclear RNA (snRNA) in the extract is eliminated by an antisense U6 DNA oligo and ribonuclease H; a "neutralizing" U6 DNA oligo was then added to protect the incoming U6 RNA. This protocol allows study of the role individual bases or the phosphate backbone of U6 plays in splicing and of the interaction between U6 snRNA and the spliceosomal proteins.

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.