Invariant U2 snrna Nucleotides Form a Stem Loop to Recognize the Intron Early in Splicing

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1 rticle Invariant snrn Nucleotides Form a Stem Loop to Recognize the Intron Early in Splicing Rhonda Perriman 1, * and Manuel res, Jr. 1 1 enter for Molecular Biology of RN, Department of Molecular, ell, and Developmental Biology, University of alifornia, Santa ruz, Santa ruz, 9064, US *orrespondence: perriman@biology.ucsc.edu DOI.16/j.molcel SUMMRY snrn-intron branchpoint pairing is a critical step in pre-mrn recognition by the splicing apparatus, but the mechanism by which these two RNs engage each other is unknown. Here, we identify a snrn structure, the branchpoint-interacting stem loop (), which presents the nucleotides that will contact the intron. We provide evidence that the forms prior to interaction with the intron and is disrupted by the DExD/H protein Prpp during engagement of the snrn with the intron. In vitro splicing complex assembly in a -destabilized mutant extract suggests that the is required at a previously unrecognized step between commitment complex and prespliceosome formation. The extreme evolutionary conservation of the suggests that it represents an ancient structural solution to the problem of intron branchpoint recognition by dynamic RN elements that must serve multiple functions at other times during splicing. INTRODUTION The spliceosome is a highly dynamic ribonucleoprotein complex in which ordered rearrangement of the spliceosomal snrns accompanies the assembly of a functional splicing complex (res and Weiser, 199; Staley and Guthrie, 1998). The large number of spliceosomal RN-RN rearrangements greatly exceeds those known for any other RN-protein complex, and their details remain poorly described. In particular, many of the most conserved snrn nucleotides have multiple functions, confounding standard analysis methods. Genetic and crosslinking studies show that snrn base pairs to the 0 splice site and pairs with the intron branchpoint sequence in an early complex in which the pre-mrn reactive groups for the first step are identified and brought together. then leaves, delivering the 0 splice site to U6 snrn. U4 snrn, having entered the complex in association with U6 snrn, also leaves, allowing U6 to pair with (res and Weiser, 199; Staley and Guthrie, 1998). s U4 departs, U6 forms an internal stem loop that promotes catalysis in part through binding a divalent metal ion (Brow, 2002). This elaborate path of rearrangements demands that many snrn nucleotides pair with more than one other nucleotide over time, and is distinctly different from the folding of the mechanistically similar group II introns (Toor et al., 2008, 2009). This additional complexity likely reflects the fact that the spliceosome acts in trans on a complex set of substrates in response to regulatory cues, whereas the typical group II intron removes itself constitutively from a single transcript. onsistent with the requirement for RN structural rearrangements, eight DExD/H box proteins play distinct and critical roles during the splicing process (Staley and Guthrie, 1998; Brow, 2002). Two of these, Prpp and Sub2p, function early during TP-dependent snrnp recruitment to the pre-mrn and stable prespliceosome formation (Kistler and Guthrie, 2001; Libri et al., 2001; O Day et al., 1996; Ruby et al., 1993; Zhang and Green, 2001). One role for Prpp is to mediate the RN structural transition from stem IIc to stem IIa (Hilliker et al., 2007; Perriman and res, 2007), a reaction antagonized by the RN-binding protein us2p (Perriman and res, 2000, 2007; Perriman et al., 2003). Stem IIa is required to form stable prespliceosomes (Yan et al., 1998; Zavanelli and res, 1991), and mutations in stem IIa are suppressed by alterations in us2p (Yan et al., 1998). dditional as yet unrecognized RN rearrangements might also be mediated by PRP (Kosowski et al., 2009; Perriman et al., 2003; Xu and Query, 2007; Yan and res, 1996). Temperature-sensitive prp alleles render lethal several mutations in invariant nucleotides that flank the -branchpoint interaction sequence (Yan and res, 1996). In addition, TPase-defective prp alleles can suppress intron branchpoint mutants (Perriman and res, 2007; Xu and Query, 2007). Strikingly, the Prpp TP-binding function, but not Prpp, is unnecessary when us2p is absent or if stem loop IIa is hyperstabilized by mutation (Perriman et al., 2003). Thus, PRP might modulate additional rearrangements involving, but the nature of these has remained obscure. Here we identify the branchpoint-interacting stem loop (), an evolutionarily conserved RN structural element that forms from pairing invariant sequences flanking the branchpoint interaction sequence. We show that this stem loop plays an important role with Prpp in coordinating the fidelity and progression of splicing during the early steps of spliceosome assembly. Mutations that destabilize the accumulate an unusual complex in vitro, whose snrn requirements and 416 Molecular ell 38, , May 14, 20 ª20 Elsevier Inc.

2 -Intron Branchpoint Recognition B Prpp Domain 1 Domain 2 N D494 prp-δ494 wt US2+ U33 U42; 46U prp-δ494-46u prp-δ494 wt PRP -46U prp-δ494-46u PRP -46U cus2δ PRP, cus2δ prp-δ494, cus2δ wt 29U U42 46U D Homo sapiens mammal Gallus gallus bird Tetraodon nigroviridis fish nolis carolinensis reptile iona intestinalis sea squirt iona savignyi sea squirt plysia californica sea slug Strongylocentrotus purpuratus urchin nopheles gambiae mosquito Drosophila virilis fly Bombyx mori moth pis mellifera bee Brugia malayi nematode aenorhabditis brenneri nematode Oryza sativa plant jellomyces capsulatus fungus spergillus fumigatus fungus Trichoderma virens fungus occidioides immitis fungus Fusarium oxysporum fungus oprinopsis cinerea mushroom ryptococcus neoformans yeast Saccharomyces kluyveri yeast Schizosaccharomyces japonicus yeast Paramecium tetraurelia ciliate Tetrahymena thermophila ciliate Trichomonas vaginalis protozoan Trypanosoma cruzi protozoan Entamoeba histolytica protozoan ryptosporidium hominis protozoan Plasmodium falciparum protozoan stem I 29 intron bp int -U6 II -U6 I -U6 III stem IIa UGU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.UUUUU.UGU UGU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.UUUUU.UGU UGU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.UUUUU.UGU UGU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.UUUUU.UGU UGU.U.G.GU.UUU...UG.UG.UGU..GU.GUGU.UGUUU.UUG.GUUGU.UGG UGU.UU.G.GU.UUUU..GG.U.GU..GU.GUGU.UUGUUU.UUG.UUUU.UGGG UGU.UU.G.G..UUUU...GG.U.GU.GU.GUGU.UUGUUU.UUG.UUUU.UGU UGU.UU.G.G..UUUU...GG.U.GUU..GU.GUGU.UUGUUU.UUUG.UUUU.UG UGU.UU.G.G..U...GG.U.GU.GU.GUGU.UUGUUU.UUG.UUU.UGU UGU.UU.G.G..UUU...GG.U.GU.GU.GUGU.UUGUUU.UUG.UUU.UGU UGU.UU.G.G..UUUU...GG.U.GU.GU.GUGU.UUGUUU.UUUG.UUUU.UG UGU.UU.G.G..UUUU...GG.U.GU.GU.GUGU.UUGUUU.UUG.UUUU.UGU UGU.UU.G.G..UUU...GG.U.GU.GU.GUGU.UUGUUU.UUG.UUUU.UGU UGU.UUUG.GU..UUU...G.U.GU.GU.GUGU.UUGUUU.UUGU.UUU.GGU.UU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.UUUUU.UGU.GU.UU.UU.G..GU...GG.U.GU..GU.GUGU.UUGUUU.UUUG.UUUUU.UG.GU.UU.UU.G..UUUU...GG.UU.GU..GU.GUGU.UUGUUU.UUUG.UUUUU.UG.GU.UU.UU.G..UUUU...GG.UU.GU..GU.GUGU.UUGUUU.UUUG.UUUUU.UG.GU.UU.UU.G.UU...UGG.UU.GU..GU.GUGU.UUGUUU.UUUG.UUUUU.UG.GU.UU.UU.G..UUUU...GG.UU.GU..GU.GUGU.UUGUUU.UUUG.UUUUU.UG..UU..G.G..UUUU...GG.UU.GU..GU.GUGU.UUGUUU.UUG.UUU.UUU UU.UU..G.G.UUUUU..GG.U.GUU..GU.GUGU.UUGUUU.UG.UGUGG.GGUU.U.UU.UU.G..UUUU...GG.UU.GU..GU.GUGU.UUGUUU.UUUG.UGU.UGG UUU.UU.UU.G..UUUU...GG.UU.GU..GU.GUGU.UUGUUU.UUUG.UUUUG.UG UU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.GUGUU.UGU UU.UU.G.G..UUUU...GG.U.GU..GU.GUGU.UUGUUU.UUG.UGUG.UGU UUG.U.GG.GG..UU...G.U.UGU..GU.GUGU.UUGUUU.GUG.UUGG.GGUU UUU.UU.G.GU..UUU...G.U.GU..GU.UUUU.UGUUU.UUG.GUUU.UGU UU.UU.G.G..UUUU..GG.UU.UGU..GU.GUGU.UUGUUU.GG.UUU.UUGU UU.UU.G.G..UUUU...GG.U.UGU..GU.GUGU.UUGUUU.UUG.UGUG.UGU.U.UU.G.G..UUU...GG.U.GUU.UU.GUGU.UGUUU.UUG.GUGUG.UGU U33 U42 46 Figure 1. Point Mutations in snrn Rescue a Large Deletion of Prpp () Prpp. The arrow shows the position of truncation (residue 494), removing the terminus and domain 2 of the conserved TPase domain. (B) Rescue of prp-d494 by -46U is cus2d dependent. Yeast disrupted for chromosomal PRP,, and US2, expressing PRP or prp-d494, Wt- or -46U, and US2 or vector on FO at for days. () Other mutations rescue prp-d494 when us2p is absent. Growth of yeast dilutions with PRP (left) or prp-d494 (right) and the indicated mutant is shown. (D) Phylogenetic comparison of the 0 end of snrn from 31 organisms demonstrates high conservation and universal conservation of suppressor residues (arrowed above). The 9 base inverted repeat (cyan) flanking the branchpoint interaction sequence (intron bp int) of the proposed is indicated. Other RN-RN interactions involving overlapping portions of RN are: U6 helix II (green), stem I (pink), U6 helix I (yellow), and U6 helix III (purple). kinetics suggest it is a product of a commitment complex that is slow or unable to convert to stable prespliceosomes. Hyperstabilized mutants relax the stringency of intron branchpoint selection, suggesting a role for the in splicing fidelity. We propose the presents nucleotides to the intron branchpoint at a critical point in spliceosome assembly. RESULTS Mutation of Invariant Residues Rescues a Lethal Truncation of Prpp To search for additional functions of Prpp in association with RN, we identified a -terminal truncation of Prpp we thought might be stable and bind TP but would not function as an TPdependent helicase. rystal structures of individual domains from several DExD/H box proteins indicate that globular domains 1 and 2 form independent stable structures in vitro, and that the majority of TP-binding site contacts reside in domain 1 (heng et al., 200; Fan et al., 2009; Rudolph et al., 2006). If a protein composed of the N terminus and domain 1 of Prpp is stable in vivo in the absence of domain 2 and the -terminal residues, it might retain a partial Prpp activity. We created prp-d494 by replacing amino acid 494 with a termination codon and removing domain 2 of the TPase domain and the terminus (see Figure 1). This deletion is unable to complement the lethal phenotype of a prp null mutation under a variety of growth conditions (Figures 1B and 1; data not shown), and recombinant Prp- D494p has no detectable TPase activity as compared to the wild-type protein in vitro (see Figure S1 available online). To determine whether a mutant RN could rescue prp- D494, we screened a mutant library (Yan and res, 1996). Because us2p is a negative regulator of Prpp function (Perriman et al., 2003; Perriman and res, 2007), we performed the screen in a cus2d strain. We isolated a single suppressor allele, -46U, which was able to support growth of prp-d494 at (Figures 1B 1D). To identify more suppressors, we Molecular ell 38, , May 14, 20 ª20 Elsevier Inc. 417

3 -Intron Branchpoint Recognition B T1 D B V1 0 T1 0 3' IIb 40 Y Y 0 Y 20 Stem loop IIa I ' G43 G37 G34 G32 G26 46 U4 U44 G43 U42 41 U40 a39 u38 g37 a36 u3 g34 U33 G G stem a loop b c stem ' a 40 U U 4 3 U U 2 c b ' * Figure 2. Structure Mapping of a Model RN () snrn folded to contain the. Nucleotides 1 92 of yeast showing stem IIa, stem IIb,, and part of stem I. The branchpoint interaction sequence is in gray. J, universally conserved pseudouridines. Nucleotides in other snrn pairings are as in Figure 1D. (B) Nuclease T1 (lanes 1,, and 6) and V1 (lanes 3 and 4) digestion of 0 end-labeled synthetic model RN. Lane 1: T1 digestion under denaturing conditions (D) with G residues indicated. Lane 2: alkaline ladder (B) with sequence indicated at left. Lanes 3 and 4: V1 digestion for 0 or min, respectively, with products indicated by gray circles. Lanes and 6: T1 digestion for 0 or min with products indicated by white circles. () V1 (arrows) and T1 (triangles) cleavages mapped to the model sequence. Note that V1 cleavage products migrate more slowly than the corresponding T1 or alkaline hydrolysis products due to the absence of a 3 0 phosphate, which reduces the negative charge-to-mass ratio of the RN. The aberrant migration is exacerbated for lower molecule weight species, where charge-to-mass ratio changes have a greater effect (uron et al., 1982). Thus, we consider assignment of the smaller V1 cleavage products to be tentative, but correct to within at least two residues. screened a second pool of snrn alleles and identified another four mutations that rescue prp-d494: -29U, -U33, -U42, and -46U; U42 (Figures 1 and 1D). These mutants grow indistinguishably from wild-type when wild-type Prpp is present, indicating that function is not greatly compromised (Figures 1B and 1; Yan and res, 1996). Each suppressor mutation alters a universally conserved residue near the branchpoint interaction region (Figure 1D). This region of RN contributes to stem I (Sashital et al., 2007), U6 helix Ia (Madhani and Guthrie, 1992), and U6 helix III function (Sun and Manley, 199). In addition, evidence from both yeast and mammalian systems indicates that conserved spliceosomal proteins interact with these nucleotides (Dybkov et al., 2006; Yan and res, 1996). Thus, these highly conserved residues play numerous roles during splicing. The distribution of suppressor mutations was inconsistent with disruption of any single known spliceosomal RN structure; however, they all fall within and disrupt a highly conserved 9 base pair imperfect inverted repeat (shaded in cyan in Figure 1D), not previously recognized as a RN structural element. duplex formed by these sequences would present the -branchpoint-interacting nucleotides (Parker et al., 1987; Zhuang and Weiner, 1989) in a terminal loop (Figure 1D, intron bp int). lthough aberrant formation of part of this structure was previously suggested to explain the growth defect of a -U44 allele (Yan and res, 1996), this stem loop has not been hypothesized to function in splicing. We have called this structure the branchpoint-interacting stem loop (). The rescue of prp-d494 by -destabilizing mutations is consistent with a role for Prpp in disrupting the during the normal process of splicing in wild-type cells. Model RN omprising Residues of Yeast Folds into a 9 Base Pair Stem Loop Of the 9 base appositions in the proposed, one is a G U pair, one is an pair, and a third is a U c pair (Figure 2). Elsewhere in the splicing machinery, non-watson-rick pairings are observed, in particular where one RN strand displaces another (Huppler et al., 2002; Massenet et al., 1999). However, because of the extreme evolutionary conservation of this sequence, phylogenetic covariation of residues that would support the existence of the stem is unavailable (see Figure 1D). To determine whether the sequence forms a stem loop, we synthesized a 27 nt model and tested its ability to fold using nuclease structure probing (Figure 2B). Nuclease V1, which cleaves bases in duplex or which are stacked (uron et al., 1982), cleaves primarily in the stem of the model (Figure 2B, lane 4, gray circles; Figure 2, black arrows). In contrast to V1, nuclease T1, which is single stranded G specific, cleaves primarily in the loop under native conditions (Figure 2B, lane 6, white circles; Figure 2, white triangles), but recognizes the stem G residues under denaturing conditions (Figure 2B, lane 1). We conclude that the highly conserved 9 bp imperfect inverted repeat sequence of yeast residues 418 Molecular ell 38, , May 14, 20 ª20 Elsevier Inc.

4 -Intron Branchpoint Recognition IIb -stem I B WT prp-δ494, cus2δ 40 Ψ Ψ Ψ 0 G IIa snrn I 20 ' 46U G26 G26; 46U U 3' Ψ U; U42 U42 IIb 40 Ψ Ψ - ; U42 0 IIa G 20 snrn ' U6-WT -WT 46U G26 G26; 46U PRP, cus2δ 3' ' U6 snrn 3' U6-8U 9U 9U; G26 -WT G26 stem I -U6 helix Ia 3' IIb IIa 0 Ψ Ψ III 40 Ψ Ia G Ib 20 snrn -U6 Helix Ia/b II ' U6-WT 46 G26; 46 G26U G26U; 46U G26U; 46 Figure 3. Genetic Evidence for Function () Structural rearrangements of snrn during splicing. snrn folded as stem I form (top), the (middle), and U6 helix Ia/b (bottom). The branchpoint interaction sequence is in gray; nucleotides 9, G26 and 46, and U42, and U6 nucleotide 8 are highlighted in white on black circles. olored sequences are as for Figure 1D. (B) Watson-rick pairing at the or 42 base pair abrogates prp-d494 suppression by non-watson-rick appositions. Growth of cus2d yeast expressing prp-d494 with indicated genes on FO, 4 days,. () G26 interacts with both 46 in the and U6-8U in U6 helix Ia. Growth of cus2d yeast expressing PRP with indicated and U6 genes on FO, 4 days, is capable of adopting the predicted structure. This experiment speaks only to the ability of the sequence to fold independently under physiological salt and temperatures, and does not incorporate adjacent spliceosomal snrns, proteins, or modified nucleotides found in the native spliceosome. Other spliceosomal RN elements have been studied at high resolution using similar model RNs (Huppler et al., 2002; Sashital et al., 2004, 2007; Stallings and Moore, 1997), and this finding (Figure 2) sets the stage for detailed structural studies of the. Suppression of the Truncated prp Mutant Requires Disruption of the ll five mutant suppressors of prp-d494 destabilize potential Watson-rick base-pairing in the stem (Figure 1). To test the idea that suppression arises from disruption, we asked whether mutations that restore potential Watson-rick pairing to either the 46U or U42 suppressor alleles (i.e., 46U; G26 or U42; U) could abrogate suppression (Figure 3B). Suppression is lost when 46U is paired with G26, a mutation that restores Watson-rick pairing. The G26 mutant has a growth Molecular ell 38, , May 14, 20 ª20 Elsevier Inc. 419

5 -Intron Branchpoint Recognition Table 1. Phenotypes of Mutation in the Base Pairs of the prp-d494 Bases Mutation Base Pair Growth Rescue G26 46 G + 46U G U + + G26 cs, ts 46 G + G26; 46U U + a G26; 46 cs, ts G26U U U G26U; 46 U + G26U; 46U U U 8 J44 U J + 8 J + J44 U cs 8; J44 + 8; J44G G cs 29 G43 G + 29U U G G + J42 J + J42 + J42G G + J ; J U U J + U; J42 U + a U U cs U33 39 U + U G26 46 J42; 46U G U ts + J42 J, pseudouracil replaces U in wild-type; +, growth;, no growth; cs, cold-sensitive growth; ts, heat-sensitive growth. a brogation of prp-d494 suppression by restoration of Watson-rick pairing. defect (Madhani and Guthrie, 1992), making evaluation of prp- D494 suppression problematic. Because the growth defect is suppressed when combined with 46U (see below), prp-d494 suppression can be evaluated in the double mutant, and it fails. Thus, restoring the base pair eliminates suppression of prp-d494 (Figure 3B). Similar results are obtained at the 42 base pair. The suppressor activity of U42 is abrogated by U, which restores Watson-rick pairing, but not by, which does not (Figure 3B). Not all nucleotide substitutions that disrupt the can suppress prp-d494 (Figure 3; Table 1), probably because nucleotide identity at these positions is heavily constrained by other functions that employ these bases. Mutations that are predicted to hyperstabilize the do not rescue prp-d494 (Table 1), and exhibit cold-sensitive or lethal phenotypes in otherwise wild-type strains (Yan and res, 1996) (Table 1). This pattern of phenotypes is consistent with the interpretation that the is a conserved dynamic structural element of snrn with a finely tuned stability, which must form and execute a function, and then be disrupted, most likely by the action of Prpp, so that subsequent steps can occur. Base Pairs Make Multiple ontributions to Splicing To this point, our test of function has involved rescue of a grossly defective Prp protein by disruption of the. To determine whether the is important for normal spliceosome function, we tested growth of mutants in strains lacking us2p but expressing wild-type Prpp. Mutation of G26 causes severe growth defects, attributed to the disruption of other snrn interactions during splicing (Madhani and Guthrie, 1992, 1994b; McPheeters and belson, 1992). G26 pairs with 9 in stem I (Figure 3, top), and also with U6 8 in U6 helix Ia (Figure 3, bottom). In the, G26 is proposed to pair with 46 (Figure 3, middle). To test whether G26 plays functional roles by pairing with 46 in the, we asked whether base-pairing between residues 26 and 46 contributes to function. 46U suppresses the G26 growth defect (Figure 3). Similarly, 46 suppresses the lethality of G26U, whereas non-watson-rick combinations do not function (Figure 3; Table 1). We conclude that interaction between bases 26 and 46 is important for splicing function, probably by forming the base of the. Because G26 participates in stem I and U6 helix Ia as well as in the (Figure 3), we tested the relative functional importance of each of the three interactions made by G26 by comparing the growth of cells lacking each of the interactions. compensatory mutation that restores stem I only (9U; G26) fails to rescue growth, arguing that disruption of stem I by G26 is not a major cause of the growth defect. In contrast, and consistent with previous reports (Madhani and Guthrie, 1992), the restoration of U6 helix Ia by U6 8U greatly improves growth compared with G26 alone. Rescue via U6 helix Ia restoration is slightly more robust than through restoration of the (Figure 3). We conclude that both the and U6 helix Ia have important functions, and that residues including G26 in the exchange with U6 helix Ia during splicing. Two nonstandard base pairs are found in the : 8 J44 and onverting either of these base pairs to Watson-rick pairs results in cold-sensitive growth, and restoring non-watson-rick pairs at these locations restores function (Table 1). old-sensitive phenotypes caused by hyperstabilization of RN structures are observed elsewhere in the spliceosome where RN structures must form and then be disrupted for splicing to progress (Fortner et al., 1994; Hilliker et al., 2007; Li and Brow, 1996; Perriman and res, 2007; Staley and Guthrie, 1999; Zavanelli et al., 1994). Taken together, the data show that the contributes to function, but must be disrupted, likely by the action of Prpp, for the necessary program of snrn rearrangements to proceed. Destabilized Mutants ccumulate an Unusual Splicing omplex In Vitro lthough the above genetic studies reveal important structural interactions between snrns at base-pair resolution, they 420 Molecular ell 38, , May 14, 20 ª20 Elsevier Inc.

6 -Intron Branchpoint Recognition wild type U42;46U (min) extract: treatment: wild type + TP TP U6 U42;46U + TP TP U6 PS/SP * 2 1 SP 2 1 PS 2 * D WT pre-mrn UU B wild type t (min) * 40 PS/SP 20 0 U42;46U t (min) PS * Figure 4. Splicing Extracts with Mutant ccumulate an Unusual omplex () Time course of splicing complex assembly at 17. RP1 pre-mrn incubated in splicing extracts from (lanes 1 ) or U42; 46U (lanes 6 ) yeast. Reactions were stopped at 0,,, 20, and 40 min after TP + pre-mrn addition, and run on native agarose-acrylamide gels. Prespliceosomes/spliceosomes (PS/SP) and commitment complexes 1 and 2 (1, 2) are identified. The unusual complex is highlighted with an asterisk. (B) Quantification of splicing complex accumulation from three independent experiments: commitment complex (), the unusual complex (*), and prespliceosome/spliceosome (PS/SP) for (left) or U42; 46U (right) are plotted as time (x axis) versus % counts in each complex (y axis). Standard deviations derive from three independent experiments. () Requirements for forming the unusual complex. (lanes 3 and 8), (lanes 4 and 9), or U6 (lanes and ) oligonucleotide ablation of (lanes 3 ) or U42; 46U (lanes 8 ) extracts was performed followed by complex formation using RP1 pre-mrn. TP depletion of (lane 2) or U42; 46U (lane 7) extracts was performed followed by complex formation using RP1 pre-mrn. omplexes are as in (). (D) Substrate branchpoint requirement for forming the unusual complex. Time course from 0 to 60 min on WT (lanes 1 6) or DUU (lanes 7 12) pre-rp1 in and U42; 46U extracts. omplexes are as in (). provide little information about the precise steps of splicing at which the might function. To explore this, we made splicing extracts from yeast strains expressing a functional but destabilized (-U42; 46U) and analyzed spliceosome assembly using radiolabeled synthetic RP1 pre-mrn as a substrate (Figure 4). U42; 46U extracts form prespliceosomes slowly, and accumulate an unusual complex (Figure 4, asterisk) that migrates between commitment complex II (2) and the prespliceosome plus spliceosomes (PS/SP) bands on the native gel (Seraphin and Rosbash, 1989) (Figure 4, lanes 6 ). This complex is not apparent in wild-type splicing extracts (Figure 4, lanes 1 ). In addition, the complex does not accumulate in hyperstabilized mutant -U44 splicing extracts (data not shown). To assess the kinetic relationship of the unusual complex with the known splicing complexes, we measured the amount of RP1 RN in each complex over time and plotted each as a percentage of total counts for each time point (Figure 4B). In comparison to wild-type (left), the U42; 46U extracts (right) show significantly slower turnover of commitment complex and production of prespliceosome and spliceosomes. The unusual complex peaks at min and slowly disappears over time. s both the commitment complex and unusual complex disappear, there is a corresponding increase in prespliceosome and spliceosomes. We suggest the unusual complex represents a kinetic intermediate whose progress along the splicing path is retarded because of either delayed formation or premature destabilization. lternatively, the complex may represent Molecular ell 38, , May 14, 20 ª20 Elsevier Inc. 421

7 -Intron Branchpoint Recognition a dead-end intermediate, the result of mis-timed pairing between and the intron branchpoint. In either case, these results support the hypothesis the helps engage with the intron branchpoint during formation of prespliceosomes. To support the conclusion that the unusual complex is related to standard splicing complexes, we determined the U snrnp, TP (Figure 4), and intron branchpoint (Figure 4D) requirements for its formation. We used snrn-specific oligodeoxynucleotides and endogenous RNaseH activity in the extracts to digest the,, and U6 snrns (lanes 8 ). When these oligonucleotides are added to control wild-type extracts, expected results (McPheeters et al., 1989; Perriman and res, 2000) are obtained, showing that is first required for commitment complex formation, is first required for prespliceosome formation, and U6 is not required until after prespliceosome formation (Figure 4, lanes 3 ). In U42; 46U splicing extracts, formation of the unusual complex requires (lane 8) and (lane 9) but not U6 (lane ) snrns, showing that it has similar snrn requirements to authentic prespliceosomes. In contrast, depletion of TP shows that the unusual complex can form in the absence of TP (lane 7), suggesting that it shares some similarity with 2, or perhaps has bypassed the TP requirement, as observed for prespliceosome formation in extracts depleted of us2p (Perriman and res, 2000) (note: these extracts contain us2p). test of substrate requirements for formation of the unusual complex (Figure 4D) shows that, like 2 (Legrain et al., 1988) and prespliceosomes (Rymond and Rosbash, 1986), it requires a pre-mrn branchpoint sequence (Figure 4D, compare lanes 1 6 with lanes 7 12). We conclude that disrupting the sequence disrupts the normal course of prespliceosome formation, resulting in accumulation of an unusual stalled complex that is hung up in the transition between commitment complexes and prespliceosomes. Hyperstabilizing the Relaxes Branchpoint Recognition Given biochemical evidence that the plays a role in stable association of snrnp with the pre-mrn and the provocative positioning of the intron contacting nucleotides in the loop of the, we hypothesized that the might play an important role in recognition of the intron branchpoint. To test this, we used T-UP1 reporter pre-mrns in which copper tolerance depends on splicing efficiency (Lesser and Guthrie, 1993). Reporters containing branchpoint region mutations (Figure ) were compared to the wild-type reporter in cells carrying different mutations on copper medium (Figure B). Wildtype T-UP pre-mrn containing a canonical branchpoint sequence shows a decrease in copper tolerance in cells expressing either the hyperstable or destabilized mutants relative to wild-type (Figure ). This demonstrates that correct stability is important for efficient splicing of wild-type premrn. In contrast, branchpoint mutations 29G and 26 (UU to UUG or UU; Figure ) demonstrate increased copper tolerance in the hyperstabilized mutant as compared to wild-type (Figure ). Hyperstabilization at position 44 is responsible for this, because disruption of the 44 8 pairing in the U44; 8 mutant eliminates both the diminished copper tolerance of the wild-type reporter and the improved copper tolerance of the mutant reporters (Figure 4D). Thus, hyperstabilizing the (U44) relaxes the stringency of branchpoint recognition, allowing mutant pre-mrns to be more efficiently spliced. onversely, destabilizing the (U42; 46U) does not allow improved splicing of mutant branchpoint introns, and appears to cause a greater decrease in tolerance to copper when compared with wild-type (Figure ). The hyperstabilizing mutation does not suppress T-UP pre-mrns mutated at the 0 or 3 0 splice sites or other mutations at the branchpoint interaction sequence (Figures S2 and S2B), demonstrating the specificity of the suppression for the intron branchpoint sequence. In contrast, destabilizing the causes a reduction in copper tolerance of the 0 splice site mutation, and more subtly for the 3 0 splice site mutant 2U, as well as for the branchpoint mutants. lthough functional interactions between,, and the 0 exon are established early in spliceosome assembly (Das et al., 2000; Donmez et al., 2004, 2007; McGrail and O Keefe, 2008), destabilizing mutants may lead to a general reduction in splicing of pre-mrn substrates of several types (Figure S2). formation is mutually exclusive with the formation of other structures, including U6 helix Ia (see Figure 3). To assess the possibility that a hyperstable competes with U6 helix Ia and that unstable helix Ia rescues branchpoint mutants, we tested strains carrying the U6 8U mutation, which replaces a G pair in U6 helix Ia with a G U pair (see Figure 3B). For the most part, cells expressing U6-8U demonstrate a similar pattern of copper tolerance for the tested premrns as with wild-type U6 (Figure ). The exception is that U6-8U on its own detectably improves splicing of the 26 branchpoint mutant with wild-type. Formally, it is not possible to tell whether this weaker rescue of 26 is due to hyperstable -mediated destabilization of helix Ia or destabilized helix Iamediated stabilization of the. However, U6-8U may act by the independent mechanism of compromising proofreading by Prp16p, as previously suggested (Madhani and Guthrie, 1994a; Mefford and Staley, 2009). Because hyperstabilization by -U44 is a more robust suppressor than U6-8U (Figure ), we conclude that relaxed branchpoint usage is due primarily to hyperstabilizing the, rather than by indirect effects of the on U6 helix Ia. To resolve and measure the effects on splicing that lead to copper tolerance, we assayed splicing by primer extension (Figures E and F; Figure S2). onsistent with the copper assays, splicing of 26 and 29G pre-mrns is improved by the hyperstabilizing mutant U44 (Figure E, lanes 3 and 6), but is greatly reduced in the destabilizing U42; 46U mutant (lanes 2 and ). Quantification (Figure F) shows the first step of splicing of both 26 and 29G pre-mrns is significantly increased (reduced fidelity of branchpoint recognition) in strains expressing U44 (Figure F) but significantly decreased in strains expressing U42; 46U. None of the three snrns improve the very poor second-step efficiency of the 29G premrn observed here and in previous studies (Burgess and Guthrie, 1993; Fouser and Friesen, 1986; Konarska et al., 2006). lthough differences in copper tolerance are observed for wildtype T-UP pre-mrn, splicing of the wild-type reporter is robust (Figure E), and quantification shows only slight changes 422 Molecular ell 38, , May 14, 20 ª20 Elsevier Inc.

8 -Intron Branchpoint Recognition Figure. Hyperstable Suppresses Branchpoint Mutations () T1-UP1 reporter pre-mrn. Point mutations 26 and 29G are shown. ircled 29 is the residue that attacks the 0 splice site for the first step of splicing to form the branch. (B) secondary structure showing U44, U42; 46U, and 8 mutations. () opper sensitivity of T-UP reporters in cells expressing wild-type U6 (upper) or U6-8U (lower) and wild-type, U42; 46U, or U44. Dilute cultures were spotted on 1.0 mm u 2+ (WT) or 0.1 mm u 2+ (29G and 26). Maximum copper-allowing growth is indicated (right of panel) with increased u 2+ tolerance circled. (D) Disrupting hyperstabilization restores copper tolerance to wild-type and abrogates branchpoint mutant suppression. opper-sensitivity assays of T- UP (WT) or 29G when Watson-rick pairing at positions is disrupted (U44; 8). Dilutions and u 2+ are as for (). (E) Splicing analysis of reporters. Primer extension products from strains expressing 26 (lanes 1 3), 29G (lanes 4 6), or wild-type (lanes 8 ) T-UP substrates. alleles are indicated at top; control strain lacks any T-UP plasmid. P, pre-mrn; M, mature mrn; L, lariat intermediate. (F) Quantification of step 1 and step 2 splicing efficiencies of 26 and 29G pre-mrn with, U44, or U42; 46U. First-step (black bars) and secondstep (white bars) efficiencies are calculated as (M + L)/(P+M+L) and M/(M+L), respectively (Query and Konarska, 2004). Standard deviations are from three biological replicates. in the appropriate direction (Figure S2B). lthough the 26 substrate appears to show a decrease in the second step with the destabilizing mutant (Figure F), the effect on the first step is so strong that the second-step efficiency cannot accurately be estimated by this method. From these data, we conclude that hyperstabilizing the relaxes the stringency of branchpoint recognition and suppresses branchpoint mutant pre-mrns at the first step of splicing, whereas destabilizing the appears to cause a general loss of efficiency of branchpoint recognition and splicing. DISUSSION The binding of snrnp to pre-mrn is an early signature event in spliceosome assembly. RN and its partner U6 possess fluid structures that are dramatically influenced by Molecular ell 38, , May 14, 20 ª20 Elsevier Inc. 423

9 -Intron Branchpoint Recognition II + snrnp hyperstable GUUGU UU -cold sensitive (slow to progress?) -decreased branchpoint stringency -abolish Prp-D494 suppression prespliceosome Figure 6. Model for Function during Spliceosome ssembly In wild-type, the helps initiate the -branchpoint interaction (left to middle). The is then disrupted, in a reaction involving Prpp, allowing stable prespliceosomes to form, leaving the stem nucleotides free to base pair with U6 snrn (middle to right). When stability is altered, splicing efficiency becomes compromised. n unstable (middle bottom) causes mis-timed branchpoint pairing, decreasing the rate of splicing, but can suppress the prp-d494 lethal phenotype. onversely, a hyperstable (middle top) decreases the stringency of intron branchpoint selection, is cold sensitive, and abolishes prp-d494 suppression. IIb + IIa GUUGU UU I formed unstable disrupted TP a program of RN-RN interactions executed during spliceosome function. In this paper, we describe a dynamic RN structural element, the branchpoint-interacting stem loop or, and place its function at the time of branchpoint recognition by the snrnp (Figure 6). The identification of this structure greatly improves our understanding of the nearly invariant RN sequences found at the core of the spliceosome. Its existence likely escaped detection because many of its nucleotides play other roles at other times in splicing. This underscores the fundamental difference between spliceosomal RNs and other structural RNs: extreme conservation is not a result of a highly constrained single function but a grand compromise that optimizes the multiple functions imposed on the same sequence against each other. We first recognized the through its antagonism of a severely truncated Prp protein. Four single-base changes in invariant nucleotides relieve this, allowing the mutant protein to support growth (Figure 1). Without phylogenetic variation to predict a stem loop, we mapped the structure of a model RN and found it forms a stem loop (Figure 2). Furthermore, compensatory mutations provide functional evidence that this stem is intrinsic to the splicing pathway. Its interaction with Prpp likely reflects a role for Prpp in unwinding the, allowing subsequent functions of its nucleotides to be executed, for example IIa IIb GUUGU UU. GUUGU UU Prpp GUUGU UU -mis-timed branchpoint pairing? -slower rate of splicing -Prp-D494 suppressor as part of U6 helix Ia (Figure 3). The provocative display of the -branchpoint-interacting nucleotides in the loop of the suggests a role in branchpoint recognition. Indeed, the is required during prespliceosome formation when the snrnp recognizes the branchpoint (Figure 4), and its stabilization allows greater flexibility in branchpoint usage (Figure ). The stability of the is tuned, as mutations that either destabilize or hyperstabilize it are deleterious (Table 1). Hyperstabilization creates coldsensitive growth but has a specific effect on the fidelity of branchpoint recognition, as hyperstable mutants suppress mutations in the conserved branchpoint sequence of the intron (Figure ). View of the RN Dynamics Involved in Prespliceosome ssembly The existence of the now explains previously reported phenotypes of RN mutations (McPheeters and belson, 1992; Yan and res, 1996). Mutations that hyperstabilize pairing are cold sensitive in vivo (Yan and res, 1996) and do not function in splicing reconstitution experiments in vitro (McPheeters and belson, 1992). In vivo structure probing of -U44, the hyperstabilizing mutant, we use in this study (Figure ) shows increased reactivity of -9, consistent with destabilization of competing stem I (Yan and res, 1996) and stabilization. Notably, U44; 8, which disrupts pairing between residues 44 and 28 (Figure ), both suppresses the cold sensitivity of U44 and reverses the 9 increased reactivity (Yan and res, 1996). residues are also important for snrnp protein-rn interactions. mutations predicted to destabilize the are synthetic lethal when combined with temperature-sensitive alleles of PRP and snrnp SF3a protein complex components PRP9, PRP11, and PRP21 (Yan and res, 1996). onsistent with this, SF3b protein components SF3b14a (Snu17p/Ist3p in yeast) (Gottschalk et al., 2001) and 424 Molecular ell 38, , May 14, 20 ª20 Elsevier Inc.

10 -Intron Branchpoint Recognition SF3a60 (Prp9p) both crosslink to sequences in purified mammalian 17S snrnps (Dybkov et al., 2006). But whether and how these proteins might function with the remain to be determined. Pairing between and the intron forms the branchpoint helix and is key to splicing progression. Splicing of pre-mrns with branchpoint mutations is improved by hyperstabilizing the (Figure ). This mechanism of suppression is likely to be distinct from that observed by directly improving base-pairing between and the intron (McPheeters et al., 1989; Parker et al., 1987; Smith et al., 2009; Zhuang and Weiner, 1989), although stabilizing the stem could allosterically stabilize base pairs between the loop and the intron. -mediated suppression promotes the first step of splicing (Figure ), making it different from suppression by alleles of PRP8 (Query and Konarska, 2004) or prp16-1 (Burgess et al., 1990; Query and Konarska, 2004), both of which aid splicing progression after the first catalytic step. We suggest that hyperstabilization reduces stringency of the step at which initial pairing of loop nucleotides is converted to an extended and more stable - branchpoint helix by prolonging or enabling an otherwise inappropriate interaction. In this view, a hyperstable allows more time or repeated attempts to pass the initial pairing check between weakly complementary branchpoint sequences, allowing mutant branchpoints to be recognized. fter initial intron recognition, a conformational change mediated by Prpp leads to disruption of the and extension of the -pre-mrn pairing, forming prespliceosomes (see Figures 4 and 6). In this model, Prpp helps identify correct -branchpoint pairing and aids stem opening, allowing extended -intron interaction and stable prespliceosomes. The formation and disruption of the is consistent with the work of Xu and Query (2007), who found that splicing of intron branchpoint mutations could be suppressed by TPasecompromised Prpp alleles, and invoked an undefined conformational change associated with formation of stable -premrn complexes. We suggest structure and dynamics embody this conformational change. In addition, the orthogonal yeast pre-mrn splicing system developed by Smith et al. (2009) speaks to the extreme flexibility in the -branchpoint helix sequence in the context of the wild-type sequence. These observations suggest the is able to present a variety of functional sequences. Fine-Tuning a Structural Feature of snrn The striking invariance of nucleotides strongly supports an ancient and integral role for this structure in the major spliceosome in all eukaryotes. Like the highly conserved internal stem loop of U6, the has few consecutive Watson-rick base pairs and includes unusual base appositions. This feature is likely important to the balanced stability of the helix with its competing structures. nother notable feature is an exceptional number of posttranscriptionally modified nucleotides. In mammalian RN, the sequence contains ten modified nucleotides (res and Weiser, 199), including three universally conserved pseudouridines (J) (Ma et al., 200; Massenet et al., 1999). lthough the specific function of these is not fully understood, a role in stabilizing spliceosomal RN elements has been demonstrated (Huppler et al., 2002; Lin and Kielkopf, 2008; Newby and Greenbaum, 2002; Sashital et al., 2007). If helix stability is affected by nucleotide modifications, the greater number of modified nucleotides in the mammalian may resolve differences in branchpoint consensus conservation between the two systems. In yeast, intron branchpoint nucleotides are highly conserved, but mammalian pre-mrns contain highly divergent branchpoint sequences (Brow, 2002). Presenting the -branchpoint interaction sequence as a loop at the end of a helix increases the chance to locate and pair with the intron branchpoint. Increasing stability via nucleotide modifications could dramatically improve the chances of recognizing highly degenerate intron branchpoints (Figure ) while still ensuring enough flexibility to unwind when required. How Do RNs Get Together? Dynamics of snrnp Binding to Its Targets via RN The predicted function has parallels in the establishment of other important RN-RN interactions. The RN primer required for olel plasmid replication is regulated by an antisense RN via initial formation of a kissing complex intermediate that is converted to a stable RN-RN interaction (esareni, 1982). Similarly, retroviruses package a diploid RN genome. The pairing occurs at the dimerization initiation site and requires passage from an unstable kissing loop intermediate through conversion to a stable duplex (Paillart et al., 1996). Further, proposed models for initiator trn/codon recognition suggest correct pairing triggers a conformational change that locks the complex into a stable state (Kolitz et al., 2009). In each case, the presentation of key interacting nucleotides nucleates the interaction and is followed by disruption and extended pairing with the target sequence in a process that mirrors our model for function. Because the RN moieties within other snrnps must bind and release a defined set of target RNs during their passage through the pre-mrn splicing cycle, some of these may be similarly structured to effectively engage their substrates. It seems likely that snrnp structure has evolved to enhance the presentation of important RN strands. Interestingly, 2 snrn, the minor spliceosome counterpart of, does not seem to form the. This apparent divergence is consistent with established differences between the two splicing pathways in early steps of spliceosome assembly (Patel and Steitz, 2003). Presenting critical sequences in loops is an energetically favorable way of initiating and promoting specific RN-RN pairing events. In addition, as befits a complex cascade of RN-RN events, the establishment of the prior structure includes presentation of the next RN segments that must be paired. In this regard, we note that disruption of the exposes the nucleotides important for formation of U6 helix I (Madhani and Guthrie, 1992) and helix III (Sun and Manley, 199), which would be expected to occur in the phases of tri-snrnp addition and spliceosome activation. EXPERIMENTL PROEDURES Yeast Strains and Plasmids Yeast strains: DS4D (Perriman et al., 2003; Figures 1, 3, and 6; Table 1), JPS3 (a gift from Jon Staley; Figures 3 and ; Hilliker et al., 2007), RP01 Molecular ell 38, , May 14, 20 ª20 Elsevier Inc. 42

11 -Intron Branchpoint Recognition (Figure 4; Yan et al., 1998), and YHM118 (Madhani and Guthrie, 1994b). (LE and HIS3) and U6 RN (TRP1) plasmids are in Perriman and res (2007) and Yan and res (1996). PRP genes are on prs314 (Perriman et al., 2003). The truncation prp-d494 was created by site-directed mutagenesis (Stratagene). T-UP reporter plasmids (Figure 4) are from Lesser and Guthrie (1993). The mutant library is from Yan and res (1996) and is randomized at positions in RN. The second suppressor screen is: G26U, 29, 29U, G32, G32U, U33, U40, U40G, 41, 41G, U42G, U42, U42, G43, U44, U4, 46, U47, U47G, 46U+U42. Model RN Structure Probing 27-mer comprising nucleotides of RN (purchased from IDT) was 0 end labeled with [g- 32 P]-rTP and polynucleotide kinase, gel purified, and suspended at 0.2 pmol/ml. One microliter of RN was incubated with 0 mm Nal, mm Tris-l (ph 7.9), mm Mgl 2, 1 mm DTT, and 0.01 units RNase T1 (mbion) or units RNase V1 (mbion) for min at 23. n alkaline hydrolysis ladder was created by mixing 0.2 pmol RN in 1 ml of water with 1 ml of 0 mm sodium carbonate (ph 9.0) and incubating at 9 for min. The G-track reference reaction was done by incubating 0.2 pmol RN in 2 ml with 3 ml of 9 M urea at 9 for min to denature the RN, chilling on ice and adding 2 ml of 0.0 units RNase T1 at for 20 min. Ethanolprecipitated samples were run on a 7 M urea, 20% polyacrylamide gel and visualized by phosphoimager. opper Tolerance and RN nalysis of Reporter Splicing JPS3 with mutant and/or U6 plasmids plus various T-UP reporter constructs was grown to mid-log phase in SD medium lacking leucine (SD leu). ultures were diluted to 600 = and ml drops were plated on a series of SD leu plates containing increasing u 2+ to 2.0 mm. Plates were assayed after 4 days at. Splicing was analyzed by primer extension using 4 mg of total RN from each strain, using primers and primer extension conditions as in Perriman and res (2007). In Vitro Splicing and Spliceosome ssembly Splicing extracts derived from yeast strain RP01 (Yan et al., 1998) were cotransformed with prs314us2 and various genes. Splicing and spliceosome assembly, TP depletion, and and U6 snrn depletions were as described (Perriman and res, 2000; Perriman et al., 2003). The oligonucleotide pairs across the branchpoint interaction region, so depletion of in U42; 46U was done using an oligonucleotide in which the base substitutions are accommodated. SUPPLEMENTL INFORMTION Supplemental Information includes two figures and can be found with this article online at doi:.16/j.molcel KNOWLEDGMENTS We thank Melissa Jurica, Grant Hartzog, Benoit habot, Megan Hall, and Jeremy Sanford and the reviewers for comments; David McPheeters for U6 plasmid; hristine Guthrie for T1-UP1 reporter constructs; and Jon Staley for JPS3. We also thank David McPheeters, Imre Barta, and John belson for discussion in the early stages of the work. This work was supported by grant GM from the National Institutes of Health to M.. Received: June 24, 2009 Revised: September, 2009 ccepted: February 16, 20 Published: May 13, 20 REFERENES res, M., Jr., and Weiser, B. (199). Rearrangement of snrn structure during assembly and function of the spliceosome. Prog. Nucleic cid Res. Mol. Biol. 0, uron, P.E., Weber, L.D., and Rich,. (1982). omparison of transfer ribonucleic acid structures using cobra venom and S1 endonucleases. Biochemistry 21, Brow, D.. 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