Requirements For U2 SnRNP Addition To Yeast Pre-mRNA - Brandeis

Nucleic Acids Research, Vol. 20, No. 16 4239efficient. An assembly scheme based on these and other studiesof the early events of yeast spliceosome assembly is shown inFigure 1. [In this communication, the designation 'spliceosomes'(and 'SP' in the figures) refers to U2 snRNP-containingcomplexes whether or not they contain U4/U6 or U5 snRNPs. ]To verify this scheme and to explore other means for generatingcommitment complexes, we 'U2-killed' the extract with acomplementary oligonucleotide and endogenous RNase H activity(21,31). The rate and extent of complex formation were comparedto those of a wild-type extract.With the U2-killed extract, there was a dramatic accumulationof commitment complexes and no spliceosome formation(Figure 2, lanes 7-12), as previously shown for extractsgenetically depleted of U2 snRNP (19). Moreover, the timecourse and extent of commitment complex formation were similarto what was observed for spliceosome formation in the wild-typeextract (lanes 1-6). The data suggest that Ul snRNP addition(commitment complex formation) is relatively slow andindependent of U2 snRNP, consistent with the previous indirectassay (21), in which commitment complexes could not be seenbut their formation was inferred from a subsequent chase intospliceosomes. The results also suggest that the rapid addition ofU2 snRNP accounts for the failure of commitment complexesto accumulate in a complete extract.Mutant Ul snRNP affects U2 snRNP addition to thepre-mRNAIf commitment complexes are the substrate for U2 snRNPaddition, extracts derived from strains containing Ul snRNAmutations might generate mutant commitment complexes to whichU2 snRNP adds poorly. To test this notion, we analyzed complexformation in extracts derived from several Ul mutant strains.The effects of these mutations had been previously characterizedin vivo; some of them had no effect whereas others had substantialeffects on splicing or growth rate (24).One mutant strain had a deletion of the entire yeast core regionof Ul snRNA (AYC; the deleted region is stippled in Figure 3A)and was previously shown to be temperature-sensitive for growth(24). Seven other mutant strains contained different Ul mutations;these included a deletion of the universally conserved A loop(ALII), a deletion of the yeast-specific helix-loop VII and VmI(AViH-VIII), and a C-to-U change at position 4 (U14U) whichparticipates in base pairing to the 5' splice site region (Figure 3A).Also included were four multiple substitutions in helix VII (HVIIml to HVII-m4) (24). With the exception of the U1-4U strain,which grows slowly at 30 C and 37 C, the other six strains growwell at both 30 C and 37 C. Extracts were prepared from thenine strains (8 mutant strains and the wild-type) after growth at30 C and assayed for spliceosome assembly in vitro.Figure 3B shows a typical spliceosome assembly gel with thedifferent extracts. Extracts from Ul mutations HVII-ml to -m4had not detremental effect on complex formation; on the contrary,they generated enhanced spliceosome levels as compared to the\(slowUi(-ATP)DccWTExtractTime (min)123U2-killed WT10 20 45 12310 20 45tz%isaISSP Lslibi1U2Pr-RA wild-type extract (lanes 4-7). Extracts from the strain containingthe U1-4U mutation showed somewhat reduced levels ofspliceosomes (lane 9 and data not shown). Extracts from the strainthat carried a deletion of the universally conserved A loop (ALII)also gave rise to reduced levels of spliceosomes (lane 3). Thesmaller deletion in the yeast core region (AVII-VIII) gave riseto an even greater reduction in spliceosomes (lane 8). All of thesestrains generated parallel reductions in commitment complexformation when assayed after inactivation of U2 snRNP activity(data not shown). Most dramatic was the AYC extract in whichthere was greatly reduced spliceosome formation and a prominentaccumulation of a complex that comigrated with CC2 (lane 2).The relative efficiency of in vitro splicing in the nine extractsparalleled the relative level of spliceosome formation (data notshown).A number of criteria indicate that the major pre-mRNAcontaining complex in the AYC extract is a AYC Ul snRNPcontaining commitment complex to which stable U2 snRNPaddition occurs poorly, even after prolonged incubation time(Figure 3C). Depending on gel conditions, its migration is similarto or indistinguishable from commitment complexes formed inwild-type extracts (Figures 3B and 4A). Like the wild-typecommitment complexes, formation of this AYC complex isindependent of both U2 snRNP and ATP, and they contain AYCUl snRNA as shown by blotting of native gels (data not shown).Also, they chase poorly to spliceosomes upon addition of wildtype extract, indicating that these complexes, rather than solublefactors in the extracts, are deficient for U2 snRNP addition (datanot shown; cf. 19,26). We conclude that the efficiency or stabilityof U2 snRNP addition depends on the nature of the Ul snRNPin the commitment complexes, consistent with a precursorproduct relationship between commitment complexes and prespliceosomes.23456789-CC2cci10 11 12fastSP--- ( ATP)Figure 1. Schematic of early events of spliceosome assembly. U1: U1 snRNP;U2 snRNP; CC: commitment complexes; SP: spliceosomes.Figure 2. Radioactively-labelled pre-mRNA substrate was incubated understandard assembly conditions (see Material and Methods) with a wild-typeminiextract to form spliceosomes (SP; lanes 1-6), or with U2-killed extract (seeMaterial and Methods) to form commitment complexes (CC1 and CC2; lanes7-12). Reactions were stopped after incubation for the various times shown abovethe lanes and analyzed on a native gel as described in Material and Methods.

,d i.4240 Nucleic Acids Research, Vol. 20, No. 16A9A - CAAU25EAW.--A.AA,;A:Aor.,0 W.AAJCB"AAA*,A'I.-:.!-.; 1'.,2JUA UJGAAGG*CL;rU,3k AAkTJkGCAACXW;,NYChr; 'o.YC. Ttl' X.Er.C:r.0 AA11w.X"GA A0XAVII-VItU CC Er U'.MIS4!.At. . . .Figure 3. (A) A few mutations of Ul snRNA are presented with the proposed secondary structure of yeast Ul snRNA. The boxed regions are deleted in ALII,AYC and AVU-VIII, respectively. The arrows indicate the single point mutations made in Ul snRNA. The numbering of nucleotides starts after the tri-methyl Gcap. (B) Spliceosome assembly with the mutant extracts under standard conditions. The mutations of the Ul gene, shown above the lanes, have been describedin (24) and/or in Figure 3A. CCl and CC2, commitment complexes; SP, spliceosomes. (C) Pre-mRNA substrate was incubated with either a wild-type extract (lanes1-6) or a AYC extract (lanes 7-12) to form spliceosomes (SP). Reactions were stopped after incubation for the various times shown above the lanes. The positionsof splicesomes and commitent complexes (CC1 and CC2) are indicated. Samples were analyzed on a native gel lacking glycerol.A few lethal Ul mutant genes had been previously examinedin vvo (24). Among hes are mutants that contain the yeast coredeletion (AYC) combined with the loop II deletion (ALIl) or withpoint mutations in loop II (L1-ml or LJI-m3) (24). We interpretedtheir lehality to indicate that the double mutant Ul snRNAs couldnot support cell viability either because they manifested anexaggerated AYC phenotype, i.e., they formed commitmentcomplexes that were even less able than the AYC commitnentcomplexes to progress to spliceosomes, or because they wereunable to form commitment complexes. Because the strainscontaining the double mutant Ul snRNPs as the sole source ofUl snRNA were not viable, we initially analyzed extracts frommerodiploid strains that contained a wild-type Ul gene (carriedby the plasmid p23; see Material and Methods) as well as a secondUl gene (wild-typeFigure 4A).or mutants as shown above each lane ofExtracts from merodiploid strains that carry the singly mutantAYC Ul gene showed a semi-dominant in vtro phenotype ascompared to their haploid 'parents'; there was a clear increasein the level of commitment complexes (compare lanes 3 and 4with lanes 1, 2 and lane 11) characteristic of the AYC strain.Extracts from merodiploid strains that carried the lethal Ul genesshowed no deviation from the wild-type pattern (compare lanes5-10 with lanes 1-2), suggesting that in vitro activity of the

Nucleic Acids Research, Vol. 20, No. 16 4241haploidmerodiploidAE-EEC) OC ExtractHSPCC2cc1LI aU ill. w91z234567 I89 10 ii 12BExtractSP CC1-tcclI12345Figure 4. In vitro assays for phenotypes associated with the lethal Ul mutations.(A) Analyses of extracts derived from merodiploid strains expressing both thewild-type Ul gene and a mutant Ul gene. Spliceosome assembly was carriedunder standard conditions and assayed on a native gel lacking glycerol. Lanes1-10 show extracts from strains harboring p23 (wild-type Ul gene on acentromere plasmid) and a mutant Ul gene on another centromere plasmid (thelatter is indicated above each lane; see 24). In the case of lanes 1 and 2, thesecond plasmid also carries the wild-type Ul gene. Extracts from two independenttransformants of each strain were prepared and analyzed in parallel (note thatthe extract in lane 4 was made from half the numbers of cells as the other extractsin this gel). Lanes 11 and 12 represent extracts derived from the control haploidstrains carrying only one Ul gene as indicated (either AYC or WT). (B) Extractswere made from merodiploid strains harboring a wild-type U 1 gene under theGAL-UAS control and a mutant Ul gene as shown above the lanes (or anotherwild-type Ul gene for lane 1), after these strains were grown in glucose-containingmedium for 16 h. Spliceosome assembly was carried out under standard conditions.SP, spliceosomes; CCl and CC2, commitment complexes.double mutant U1 snRNPs may be qualitatively different from thatof the AYC Ul snRNP, consistent with the inability of the formerto support detectable growth at all temperatures tested (24).To show more directly that the Ul snRNPs expressed fromthe lethal Ul genes were not functional, several additionalmerodiploid strains were constructed and examined. Thesecontained a wild-type Ul gene under control of the GAL-UAS(provided by pXL46, see Material and Methods) as well asanother Ul gene (wild-type or mutant) with a normal Ulpromoter. To extinguish expression of the wild-type U1 snRNA,the carbon source was shifted from the galactose to glucose. Evenwith a lethal Ul mutation, the cells continue growing normallyfor 10-16 h (utilizing the previously synthesized, stable wildtype Ul snRNPs for splicing), during which time they dilutesubstantially the wild-type snRNPs and continue to synthesizethe mutant snRNPs. After 16 h, the wild-type Ul snRNAexpressed from the GAL-U1WT gene was essentially depletedby this treatment (19,24), and these extracts contain a singlepopulation ( 95 %) of Ul snRNP expressed from the other Ulgene with the conventional U1 promoter. This provides an invivo method to assemble even lethal Ul snRNA mutants into UlsnRNPs that can then be assayed in vitro in the absence of thewild-type Ul snRNP.As predicted, the phenotypes of the GAL-depleted extractsfrom strains that carried viable Ul mutants were the same asthose obtained from haploid strains that contained thecorresponding mutant Ul genes as the sole source of Ul RNA(Figure 4B). The wild-type (WT) pattern (lane 1) was similarto that from a wild-type strain (e.g., Figure 3B, lane 1). Thepattern from the viable double mutant combination AYC L126A[the AYC deletion and the L1126A point mutation; LII26A wasformerly LH-27A in (24)] resembled that of a AYC extract(compare Figure 4B, lane 4 with Figure 3B, lane 2) as well asthat from a haploid strain that carried the same double mutantcombination as the sole source of Ul snRNA (data not shown).The assembly pattern from another GAL-depleted extract thatcontained a different viable Ul gene, ALII AVII-VIH, gave riseto only a small amount of complex formation (Figure 4B, lane5), indistinguishable from what was observed with thecorresponding, viable haploid strain (data not shown). Theextracts containing the lethal combination of the yeast coredeletion and the loop H deletion (AYC ALH) formed neitherspliceosomes nor commitment complexes (Figure 4B, lane 2).Another lethal combination, AYC LII29A [see Figure 3A;L1129A was formerly LII-30A in (24)], almost completelyelimnated the spliceosome signals; there was a faint commitmentcomplex signal reminiscent of the AYC extract phenotype. Weconclude that the GAL-depletion approach can address the in vitrophenotype of lethal Ul mutations and that the lethal combinationof AYC ALII is unable to form splicing complexes in vitro.Commitment complex formation is an ATP-independent stepPrevious observations indicated that commitment complexformation does not require added ATP, in contrast to U2 snRNPaddition (19,21). To show that this is likely to be a qualitativerather than a quantitative difference, i.e., that commitmentcomplex accumulation does not depend on the presence ofendogenous ATP in the extract, we adopted a strategy to depleteATP from the extract. The protocol exploits the hydrolysis ofATP by hexokinase, which converts glucose to glucose6-phosphate (32). After first preincubating splicing extracts withhexokinase as well as glucose, we found that the addition of only

4242 Nucleic Acids Research, Vol. 20, No. 16ATP'2.0MlmrnMi 5,,4a f V, ,11 #' fFigure 5. Pre-mRNA substrate was incubated under standard conditions in a wildspheroplast extract (see Material and Methods) with various amounts ofglucose in the absence (lanes 1-3) or the presence (lanes 4-6) of 2 mM ATP.(Note that lane numbers correspond to identical samples between panels A andB; lane 2 is omitted from panel A.) Splicing intermediates (L, lariat intermediate;I, intron lariat) and unspliced pre-mRNA (P) were assayed on a 15%polyacrylamide denaturing gel (panel A), while commitment complexes (CC1and CC2) and spliceosomes (SP) were assayed on a native gel (panel B).*ttypeglucose had identical effects (data not shown and see below),due presumably to the presence of substantial hexokinase in thesplicing extracts.Addition of 1 mM glucose to the extracts completely inhibitedsplicing, even in the presence of 2 mM ATP, which wouldnormally support substantial activity (Figure 5A, lane 6). Similarresults were observed for spliceosome formation, as the additionof glucose inhibited U2 snRNP addition and led to theaccumulation of commitment complexes (Figure 5B, lanes 1-3).As expected, the addition of a sufficient excess of ATP overcamethe inhibitory effect of glucose (Figure 5B, lane 5 vs. lane 2).The results show that the small amount of spliceosome formationobserved without ATP addition (Figure SB, lane 1) is due toresidual ATP in the extract, that commitment complex formationdoes not require ATP, and that spliceosome formation requiresATP.Hydrolyzable ATP analogs promote U2 snRNP additionWe tested the ability of a dozen nucleotides (including 5 ATPanalogs) to promote spliceosome assembly and splicing afterpreincubation of the extract in 0.2 mM glucose to depleteendogenous ATP (see Material and Methods). With the exceptionof the two non-hydrolyzable ATP analogs AMP-PCP and AMPPNP (Figure 6A, lanes 13 and 14), all of the nucleotides testedwere able to effect spliceosome formation (Figure 6A, lanes2-12). To test whether this was due to the direct utilization ofthese molecules by splicing factors or to phosphate transferenzymes, we separated the whole cell extract into a snRNP anda non-snRNP fraction by ultracentrifugation (26; see Materialand Methods). The ability of the nucleotides to promotespliceosome assembly in the snRNP fraction was then examined(Figure 6B). In this case, we also observed spliceosome formationwith ATP and hydrolyzable ATP analogs (dATP and 3' dATP)Sihb*SSbIhb5 6 1sSsFigure 6. The wild-type extract (Figure 5A) and the pellet fraction of the sameextract (Figure SB) (see Material and Methods) were preincubated in the presenceof splicing salts and 0.2 mM glucose to deplete the endogenous ATP. Spliceosomeassembly was assayed with the subsequent addition of pre-mRNA and 2 mMNTP, dNTP, or ATP analog as shown above each lane (see Material and Methodsfor abbreviations). Commitment complexes (CCI and CC2) and spliceosomes(SP) were assayed on native gels.and with two poorly hydrolyzable ATP analogs (ATP-a-S, andATP--y-S) (Figure 6B, lanes 2, 6, 10, 11, and 12, respectively).However, in contrast to the results obtained with the wholeextract, all other ribonucleoside- and deoxyribonucleoside-5'triphosphates were unable to promote spliceosome assembly inthe pellet fraction, suggesting that some nucleoside diphosphatekinase-like activity did not fractionate with the active snRNPsand was responsible for the activity of these triphosphates inwhole extract.U2 snRNP is not stably associated with the commitmentcomplexes in the absence of ATPThe ATP requirement for pre-spliceosome formation cannotexclude the possibility that a U2 snRNP-containing splicingcomplex is formed without ATP but is unstable during thesubsequent assay, for example, during gel electrophoresis. Bythis hypothesis, U2 snRNP addition would be ATP-independentand ATP would be necessary to stabilize a loose interactionbetween U2 snRNP and other components of this pre-spliceosomecomplex (cf. 33-35).To address this possibility, we devised an experiment thatemployed two distinguishable U2 snRNAs, encoded by a wild-

Nucleic Acids Research, Vol. 20, No. 16 4243Extract 1Extract 2WTU2 H170 U2 -WT H170 WT H170H170 WT H170 WT%WT H170WT H170ExtractyeastRNAGIc (mM)WT U1-4Um1 2 2WT U1-4U122456uSPOLIGO 23T-1234567CC2 ccl8Figure 7. Analysis of U2 snRNA species coprecipitated with biotinylated premRNA by streptavidin-agarose. Assembly reactions (lacking added ATP) werecarried out in the presence of glucose in various extracts ['Extract 1', as shownabove lanes 1-7; wild-type (WT), HI70 or a mixture (WT H170)]. Biotinylatedpre-mRNA was the substrate for all reactions (except that in lane 6, for whichnon-biotinylated pre-mRNA was used). After 30 min, a 15-fold excess of nonbiotinylated pre-mRNA was added, followed (after 1 min) by the simultaneousaddition of ATP and another assembly reaction done with non-biotinylated premRNA in another extract ('Extract 2', as shown above lanes 1-7). After 5 min,the reactions were quenched and the complexes purified with streptavidin-agarose.For lanes 3 and 4, the reactions were mixed after incubation with ATP andquenching; for lane 7, no ATP was added (see Material and Methods for furtherdetails). U2 snRNAs in the complexes bound to streptavidin were purified andassayed by primer extension with oligo 23T and ddATP; the products wereanalyzed on a 15 % polyacrylamide den

Requirements for U2snRNPaddition to yeast pre-mRNA Xiaoling C.Liao, Hildur V.Colot, YueWang and Michael Rosbash* Howard Hughes Medical Institute, Department of Biology, Brandeis . Department of Biochemistry, Brandeis University, Waltham, MA02254, USA \11--D 1992 Oxford University Press. 4238 Nucleic Acids Research, Vol. 20, No. 16 MATERIAL .