U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

Abstract

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...

The biochemical defects ofprp4-1andprp6-1yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP

“…No free U6 snRNP was found and very little amounts of U4/U6 snRNP and [U4/U6.U5] tri-snRNP were detected in this extract (compare fractions 6-8, 12-14 and 24-28 in Figures 7C and D, respectively). In contrast, the free U5 snRNP amount (fractions [16][17][18][19][20][21][22] was increased and a free U4 snRNP was found (fractions [8][9][10][11][12][13][14]. This particle has a reduced velocity compared to the U4/U6 snRNP and very probably derives from U4/U6 snRNP disruption.…”

“…Then, the simultaneous addition of the U4/U6 and the U5 snRNPs, probably as a tri-snRNP, leads to the formation of the spliceosome. In this complex, the U4/U6 base pairing is disrupted leading to an active structure that catalyzes the splicing reaction per se (12). In addition to the polypeptides found in purified snRNPs, proteins which are not integral components of these particles are also required for efficient splicing.…”

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…A yeast strain carrying the prp2-1 mutation 72 and a C-terminally TAP-tagged Snu17p was created as described in ref. 73.…”

Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants

“…To determine if some of the differences noted above between plant and human U6atac snRNAs were functionally silent, we tested them in the context of an in vivo suppression assay for U6atac snRNA+ We have previously shown that human U6atac snRNA compensatory mutants expressed in Chinese hamster ovary (CHO) cells can suppress the in vivo cryptic splicing phenotype of 59 splice site mutants of a U12-dependent intron (Incorvaia & Padgett, 1998)+ Starting with this suppressor snRNA, we tested the effect of the A-to-G difference at position 26 of plant U6atac+ This position appears to be homologous to the invariant A of the AGC motif found in U6 snRNAs (Brow & Guthrie, 1988) and in human U6atac snRNAs (Tarn & Steitz, 1996b)+ In yeast U6 snRNA, mutation of this position leads to defects in splicing, particularly in the second step (Fabrizio & Abelson, 1990;Madhani et al+, 1990)+ However, mutation of this residue in mammalian U6 snRNA had no effect using an in vivo suppression assay (Datta & Weiner, 1993)+ When the A 26-to-G mutation was introduced into the suppressor U6atac construct (Figs+ 8A and 9), full in vivo suppressor activity was maintained showing that G 26 is fully compatible with active U12-dependent splicing+ Note that both A 26 and G 26 can potentially base pair to U12 snRNA in slightly different registers (Figs+ 7 and 8A)+ Immediately following this (A/G)GC sequence is a region that can form an intramolecular stem-loop that is similar in size, position, and structure to a critical region of U6 snRNA+ Although the plant and human U6atac sequences differ by almost 50% in this region, the predicted structures are similar+ To demonstrate that the plant stem-loop could still be active in spite of these differences, we constructed a chimeric U6atac in which the plant stem-loop replaced the human stemloop+ The resulting construct was tested for activity in vivo using the same suppressor assay described above+ We found that, in the presence of a mutated human U4atac snRNA, this chimeric U6atac snRNA was active in vivo (Figs+ 8A and 9)+ This shows that the function of the plant intramolecular stem-loop structure is conserved+ These data also provide the first in vivo evidence for the predicted function of U4atac snRNA in U12-dependent splicing+ When Tarn and Steitz (1996b) identified U6atac and U4atac snRNAs in human nuclear extracts, they noted that the two snRNAs could adopt a base-paired structure analogous to that formed by U4 and U6 snRNAs+ In the case of U4/U6 snRNA, this structure appears to be required for splicing in vivo and in vitro (Wolff & Bindereif, 1992)+ The precise role of this structure is still unclear but it has been proposed that U4 snRNA acts as a chaperone to deliver the U6 snRNA to the nascent spliceosome in an inactive form (Guthrie & Patterson, 1988)+ Subsequently, through the action of ATP-dependent helicases (Raghunathan & Guthrie, 1998), the two snRNAs are separated and U6 goes on to form alternative base-pairing interactions with U2 and the intron 59 splice site whereas U4 appears to be destabilized from the spliceosome (Lamond et al+, 1988;Yean &...…”