Splicing function of mammalian U6 small nuclear RNA: Conserved

Transcription

1 Proc. Nati. Acad. Sci. SA Vol. 91, pp , February 1994 Biochemistry Splicing function of mammalian 6 small nuclear RNA: Conserved positions in central domain and helix I are essential during the first and second step of pre-mrna splicing THORSTEN WOLFF, RTH MENSSEN, JRGEN HAMMEL, AND ALBRECHT BINDEREIF* Max-Planck-Institut far Molekulare Genetik, Otto-Warburg-Laboratorium, Ihnestrasse 73, D Berlin (Dahlem), Federal Republic of Germany Communicated by Christine Guthrie, October 12, 1993 (received for review August 10, 1993) ABSTRACT On the basis of mutational analyses in yeast, the highly conserved ACAGAGA sequence of 6 small nuclear RNA (snrna) and the adjacent 6-2 helix I have been proposed to be part of the active center of the spliceosome. We report here a detailed analysis of the human 6 snrna sequence requirements during the first and second step of splicing, using a mammalian in vitro splicing-complementation system and a mutational approach. Positions A53G"4C-" (helix Ib) were identified as important specifically for the first step, but not for spliceosome assembly. A45 of the ACAGAGA sequence and 52 of helix Ia function during the second step; in addition, the bulge separating helices Ia and lb appears critical for the second step. In contrast, no splicing-essential sequences could be identified in the central domain upstream of the ACAGAGA sequence. In sum, our data demonstrate for the mammalian splicing system that discrete positions within the ACAGAGA sequence and helix I of 6 snrna function during the first and second step of splicing, suggesting that these two sequence elements are closely associated with the catalytic center of the spliceosome. Comparison with previous results in yeast indicates a fundamental conservation of the 6 snrna function in the pre-mrna splicing mechanism. Nuclear pre-mrna splicing involves two sequential transesterification reactions, resulting in mature mrna and the intron lariat (for review, see refs. 1-3). Before the two steps of splicing, the pre-mrna has to be assembled into a highly complex ribonucleoprotein structure, the spliceosome (for review, see ref. 4). Spliceosome assembly occurs through an ordered, multistep pathway requiring many splicing factors, among them four small nuclear ribonucleoproteins (1, 2, 4/6, and 5 snrnps). Nuclear pre-mrna splicing and autocatalytic self-splicing processes are mechanistically similar, which has led to the hypothesis that they are evolutionarly related (5, 6). Specifically, the small nuclear RNA (snrna) components of the spliceosome may have functionally replaced conserved intron structures of groups I and II self-splicing RNAs (for review, see ref. 7). A strong candidate for a catalytically active component of the spliceosome is 6 RNA, which is the most conserved snrna (8). An unusual feature of 6 is that it undergoes several conformational transitions during the spliceosome cycle, including a singular form of 6 (9), a 4-6 basepaired structure (9, 10), and a spliceosomal 6-2 conformation (11). The latter conformation contains helix I, where the stem I region of 6 and a sequence near the 5' end of 2 RNA interact (11); in this context the 6 stem II region can refold into an intramolecular stem-loop (12); an additional 6-2 interaction (helix II) forms between sequences near the 3' end of 6 and the 5' end of 2 (13, 14). The current model of the active spliceosome implies that the 6-2 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18.S.C solely to indicate this fact. structure is closely associated with the catalytic center, based on the following evidence: (i) Mutational analyses in yeast have shown that 6 and 2 nucleotides in helix I, as well as the adjacent ACAGAGA sequence of 6, are functionally important in the first and second step ofthe splicing reaction; significantly, this region is immediately adjacent to the branch-point interaction sequence of 2 RNA (11, 15-17); (ii) two genetic studies in mammalian cells have shown that the 6-2 helix II is essential for splicing (13, 14), most likely at the level of spliceosome assembly or stability (18); (iii) the intramolecular helix of 6 between helices I and II is essential during the first step of the splicing reaction (12). Additional support for a catalytic role of6 RNA comes from the discovery of introns in some fungal 6 genes (19). In the present study we have used a mammalian splicingcomplementation system to determine the 6 sequence requirements during splicing, focusing on the central domain, including the ACAGAGA sequence, and helix I. Our results establish that in the mammalian system specific 6 nucleotides in helix I are required during both the first and second steps of splicing and that position A45 of the ACAGAGA box is critical for the second step; in contrast, no essential sequences could be identified further upstream in the central domain of 6. In sum, our data suggest that an extended catalytic core exists in the mammalian spliceosome that includes the ACAGAGA box and the 6-2 helix I. MATERIALS AND METHODS Assays of Splicing Complementation, Spliceosome Assembly, and 4-6 Interaction. The preparation of 4/6 sn- RNP-depleted HeLa nuclear extract and the affinity purification of 4 snrnp have been described (18). Complementation of splicing activity and spliceosome assembly by T7 RNA polymerase transcribed 6 RNAs has been analyzed, as described; 4-6 interaction has been determined by anti-sm immunoprecipitation (12, 18). Mutagenesis, in vitro Transcription, and 3'-End-Labeling. All mutant plasmids listed below were generated by amplifying mutant 6 genes through PCR methods, using T7-6 (12) as a template and mutagenic DNA oligonucleotides, followed by subcloning into pc19. In the central domain and helix I region, the following T7-6 derivatives were constructed, each carrying a 3- or 4-nt substitution (sub, as indicated by the nucleotide positions), in which the wild-type sequence is replaced by the complementary sequence: T7-6 sub 20-22, T7-6 sub 23-25, T7-6 sub 26-28, T7-6 sub 29-31, T7-6 sub 32-34, T7-6 sub 35-37, T7-6 sub 38-40, T7-6 sub (called ACA -- G in Fig. 2), T7-6 sub (called GAGA-+ CC in Fig. 2), T7-6 sub (called GA -- CA in Fig. 3), T7-6 sub (called GA -- CAA in Fig. 3), T7-6 sub Abbreviations: snrna, small nuclear RNA; snrnp, small nuclear ribonucleoprotein. *To whom reprint requests should be addressed. 903

2 904 Biochemistry: Wolff et al. In the ACAGAGA sequence and the helix I region, the following single-point mutants have been constructed: T7-6 A41'- G, T7-6 A41-., T7-6 A41-- C, T7-6 C42 -* G, T7-6 C42-3 A, T7-6 A43 -), T7-6 A43 -> C, T7-6 G44- A, T7-6 G44 -, T7-6 G44 - C, T7-6 A45-3 G, T7-6 A45 --, T7-6 A45 --+C, T7-6 G46-- A, T7-6 G46-, T7-6 G46-- C, T7-6 A47-- G, T7-6 A47 -->, T * G, T A, T * C, T7-6 A53-- G, T7-6A53-+, T7-6 A53 >C, T7-6 G54-- A, T7-6 G54, T7-6 G54 --+C, T7-6 C55 -G. T7-6 C55 - A, T7-6 C55 - C. In addition, a deletion derivative, T7-6 AA456, and a derivative with two uridine residues inserted (ins) between positions 52 and A53, T7-6 -ins, have been constructed. The sequence of each construct was confirmed by dideoxynucleotide chain-termination sequencing. All plasmids described above were linearized with Dra I and transcribed by T7 RNA polymerase in the presence of GpppG cap analogue. Synthetic T7-6 RNAs carry no extra nucleotides at their ends. T7-6 RNAs were labeled at the 3' end using [32P]pCp and T4 RNA ligase (20). MINX pre-mrna was obtained by in vitro transcription of BamHI-cut SP6-MINX (21) with SP6 RNA polymerase in the presence of m7gpppg cap analogue, as described (22). RESLTS Mutational Analysis of the Central Domain of Human 6 RNA: The Conserved ACAGAGA Sequence Is Required for Both the First and Second Step of Splidug. Recent crosslinking studies have provided evidence for an interaction between the 5' splice-site region of the pre-mrna and the central domain of 6 RNA (23-25). To define functionally important sequences in the central domain of human 6 RNA, we constructed a series of contiguous substitution derivatives covering the entire central domain (nt 20-47); in each of the 6 mutant RNAs 3 or 4 nt have been replaced by the complementary bases (for summary, see Fig. 5). These mutant RNAs were tested in splicing reactions containing 32P-labeled pre-mrna, using complementation of 4/6- depleted nuclear extract by 6 mutant RNA and purified 4 snrnp. Fig. 1 shows that seven substitutions covering nt were active in splicing complementation: Substitutions of nt 20-22, 29-31, and moderately reduced splicing activity, whereas the others were as active as wild-type 6 RNA. In contrast, two substitutions in the highly conserved ACAGAGA sequence (nt and 44-47) gave a very strong splicing defect or no detectable activity (Fig. 2, lanes ACA -. G and GAGA -. CC, respectively). In sum, these data show that the splicing function of 6 requires the conserved ACAGAGA box; upstream of ACAGAGA, however, no essential sequence element could be identified in the central domain. Next, the functional importance and contribution of single nucleotides in the ACAGAGA sequence (nt 41-47) were studied in detail. Most of the possible single-point mutations were introduced, and the resulting mutant 6 RNAs were assayed for splicing complementation (Fig. 2; summarized in Fig. 5). Many of these point mutations had no significant effect on splicing activity (activity between 50 and 100%1 of wild-type; Fig. 2, lanes A4' -l G, A4' -*, A4' -l C, C42 G, A43 -., A43-* C, G44-., A45-* G, G46-*, G46 C), several of them reduced splicing activity to levels of between 10 and 50% of wild-type (Fig. 2, lanes C42 -) A, G44 -- A, G44-3 C, G46-+ A, A47--* ); a strong first-step effect was observed only at position A47, with <10% of splicing activity remaining for the A47-- G mutation (Fig. 2, lane A47 -- G). Two very clear second-step splicing defects were identified, both at position A45: Changing A45 to either uridine or cytidine led to a strong or moderate accumulation of splicing intermediates, respectively, whereas the A45 -- G Proc. Natl. Acad. Sci. SA 91 (1994) FIG O..S 3 s o m ' (A =l L/)./ Jo LA -:---- _ -- FIG. 1. Splicing-complementation activities of 17-6 mutant RNAs with 3- or 4-nt substitutions in the central domain and the helix I region. nlabeled 17-6 RNAs were incubated with purified 4 snrnp and 32P-labeled MINX pre-mrna for 90 min in 4/6- depleted nuclear extract under splicing complementation conditions. RNA was isolated and analyzed by denaturing acrylamide gel electrophoresis. Wild-type (WI) and mutant 6 RNAs with the indicated nucleotide positions substituted by the complementary base are marked above lanes. In control reactions, splicing activity was determined in mock-depleted nuclear extract (NE), in 4/6- depleted extract (-), or in 4/6-depleted extract with 4 snrnp (4) or T7-6 wild-type RNA (6) added. Positions of the premrna, splicing intermediates, and products are indicated at right. mutation slightly reduced the first step (Fig. 2, lanes A45, A45 -* C, A45 -- G). In addition to splicing complementation, spliceosome assembly was determined for the point mutants in the GAGA sequence (nt 44-47), where the more severe splicing defects had been observed: These mutant 6 RNAs were integrated into B-type splicing complexes similarly as wild-type 6 RNA (data not shown), indicating that the first-step splicing requirement occurs during splicing catalysis, at a step past the assembly of a stable spliceosome. Taken together, our results demonstrate a requirement of the ACAGAGA sequence during the first and second step of splicing; in particular, position A45 is critical for the second step. Z_ DD<_2A 22AC =G~~~~~~~~~~~~L A... <~~~~~~~~~~~~~~L i FIG. 2. Splicing-complementation activities of T7-6 mutant RNAs with point mutations in the ACAGAGA sequence (nt of human 6 RNA). Splicing activities were determined as described in the legend to Fig. 1. Wild-type (WT) and mutant 6 RNAs are indicated above lanes. Positions of the pre-mrna, splicing intermediates, and products are indicated at right. NE, nuclear extract.

3 * An :;IIL-a; XWs Biochemistry: Wolff et al. Proc. Natl. Acad. Sci. SA 91 (1994) 905 deleted (T7-6 AA56), and (ii) two uridine nucleotides were Li inserted between helices Ta and lb (T7-6 -ins), thereby ~ =3 G 4e52 A C GA53 C GC55 A ad a L allowing a potential contiguous 6-2 helix Ta-Ib to form A C without the 2-nt bulge (A23A24 in human 2 RNA) (summa-. nrized - in Fig. 5). * -@-1g1111!! _ - - For the 3-nt substitution in helix Ia (GA -* CA) we found only a weak reduction of splicing activity, but no specific effect on the second step, suggesting that the secondstep effect seen for the 4-nt substitution (GA -+ CAA; called sub in Fig. 1) results from mutation of position - [ -I-_ 52 (Fig. 3, compare lanes GA -3 CA and GA - CAA). Significantly, point mutations at the two adjoining positions of helices Ta and Tb, 52 and A53 clearly showed base-specific splicing defects. The G substitution partially blocked the second step, whereas the 52 -* A and 52 -* C mutations had wild-type activity (Fig. 3, lanes 52 -* G, 52 -* A, 52 + C). Each of the three helix Tb intermediates, and products are indicated between the panels. NE, very low levels (Fig. 3, lanes G54 -- A, G54 -_,.G54 -- C). nuclear extract. Finally, T7-6 C55-4 G RNA complemented splicing marginally above background, whereas the C55 -- A and C55 -* Helix I Positions of 6 RNA Function During both Splicing mutations resulted in intermediate levels of activity (Fig. Steps. In the current model of 6 interactions in the splice- 3,lnsC5-*G 55-+AC5 ).Adetoofpiin osome, the 6_ helix I is located directly downstream.of of A56 3, lanes between C55 > helix G., C55 I and -> the A, C55 -+). A deletion of intramolecular 6 helix position the ACAGAGA sequence (11; see Fig. 5). Two mutant.... reduced derivatives were constructed, in which the two halves of splicingactivtyon sltly fig. 3le A very helix I (Ta, nt 49-52, and Tb, nt 53-55) had been substituted nterestnig phenotype resulted from the insertion beby the complementary sequence. These mutations resulted splicing was completely blocked, and splicing intermediates differential effects on the first and second step of splicing: strongly accumulated (Fig. 3, lane -ins). This result Splicing complementation by T7-6 sub RNA led to suggests that either the spacing between helices Ta and Tb, the an accumulation of splicing intermediates, indicating a spe- 6-2 helix I stability, or a combination of these factors is cific second-step defect ofthis mutant derivative; in contrast, critical for the second step of the splicing reaction. T7-6 sub RNA was reduced already in first-step To assess whether the splicing defects detected for helix I splicing complementation (Fig. 1, lanes sub 49-52, sub 53- mutations are, in fact, due to specific requirements during the 55). These splicing defects were further dissected by con- catalytic steps ofthe splicing reaction, we assayed each of the structing a smaller substitution derivative covering nt mutant 6 RNAs for their ability to assemble into B-type (T7-6 GA CA) and all possible single-point mutations splicing complexes. Fig. 4 shows that 6 RNAs with a GA in the AGC sequence (nt 52-55). In addition, two other -4 CA substitution or with any of the single-point mutations mutations were introduced and tested: (i) the single position were incorporated into spliceosomes, except for A53 -) separating helix I from the intramolecular 6 helix was (see below). In sum, both helix Ta and Tb are required for GA WT CA 52G 52A 52C A53G A53 A53C 654A G54 G54C C55G C55A C B ~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ *b- B-IiII*BI*II II i.h hiiaa *o^s^ s ^ ^^^v ^ s - se-s.q q....- I.'Ls' _- s:#tg He FIG. 4. Spliceosome assembly of T7-6 mutant RNAs with point mutations in helix I (nt of human 6 RNA). 32P-labeled 6 RNA.Z.s.ss.Z...s iz... X:s. l.....s ~~~~~~~~nucleotides (A53G54C55) was found important for the first FIG. 3. Splicing-complementation activities of T7-6 mutant.. RNAs with point mutations in the helix I region (nt of human splicing step. The A53 -) mutation strongly reduced 6 RNA). Splicing activities were determined as described in the splicing to levels of <10%, and A53 C reduced it to legend to Fig. 1. Wild-type (WT) and mutant 6 RNAs are indicated intermediate levels; in contrast, A53 G did not affect above lanes. The 6 mutant RNA -ins carries an insertion of two splicing activity (Fig. 3, lanes A53 -- G, A53 -*, A53 -> C). uridines between 52 and A53. Positions of the pre-mrna, splicing The G54 -- A,, or C changes reduced splicing activity to derivatives were incubated for 40 min under splicing-complementation conditions with purified 4 snrnp in 4/6-depleted nuclear extract in the presence (+) or absence (-) of 75 ng of unlabeled MINX pre-mrna. Wild-type (WT) and mutant 6 RNAs are indicated above lanes. The assembly of 6 RNAs into heparin-resistant B-type splicing complexes (B) was assayed by nondenaturing gel electrophoresis on composite acrylamide-agarose ribonucleoprotein gels (26).

4 nftc Biochemistry: Wolff et al. Proc. Natl. Acad. Sci. SA 91 (1994) A41 C42 A43 G44 A45 G46 A47 52 A53 G54 C55 G nd _- ++ 4M A 0 - a1 1&,* C C; C' ++ nd A.70 c G Intramolecular C m++ nd ++ Helix 6 c 60.C G CL- CG A Helix 11 u ins "."/A -- A CGCAAACGGMGCGCCAA + + 'lx C! / _ \G G. C(.JLJCC" GGCLCLkJi-G("~p CAAACAAAAGGAACGAACAGAGMG A X A o ~ o 4~ ~,6 A Ao 3oA a 40 GAGA - Exon 2]CGAY -----AC I ACA----- s pre --- rnrna iexon 1iG FiG. 5. Secondary-structure model of the human 6-2 interaction (modified from ref. 11) and summary of splicing complementation activities of T7-6 RNAs with mutations in the central domain and the helix I region of 6. The relevant nucleotide sequences of 6 and 2 RNAs are shown; the remainder is drawn by lines. The 6-2 helices I and II, the intramolecular 6 helix, and the base-pairing interaction between 2 and the pre-mrna branch-point region are indicated. Three- and 4-nt substitutions as well as an insertion (-ins) and a deletion derivative (AA56) are marked by a line above the sequence, together with their splicing-complementation activities. The effects of single-point mutations on splicing complementation in the ACAGAGA sequence and in helix I are summarized on top of the secondary-structure model. nd, Not determined. Splicing-complementation efficiencies are expressed in comparison with wild-type 17-6 RNA (+ +, o; +, 10-50%; +/-, <10%; -, background level; the lariat intermediate symbolizes a partial or complete block of the second step of splicing). splicing catalysis, specifically nucleotide 52 during the second step and each of the three helix Ib nucleotides (A53G54C55) for the first splicing step. The spliceosome-assembly defect of T7-6 A53 -_+ RNA could be traced back to its inability to interact with the 4 snrnp: The A53 -- mutation abolished 4-6 interaction almost completely, whereas another base substitution at the same position (A53 G) did not affect 4/6 snrnp assembly (data not shown). In the current singular 6 structure (9) this position resides in an internal bulge, which has recently been proposed to contain additional Watson- Crick base pairing (A. Mougin, R. Lthrmann, and C. Branlant, personal communication). Significantly, the A53 -. mutation would create a longer, contiguous helical region of 5 bp (nt and 80-84). The expected stabilization of the singular form of 6 may, therefore, explain why this point mutation precluded 4-6 interaction. This result indicates that the overall stability of the singular form of 6 is important for 4/6 snrnp assembly. DISCSSION We have used an in vitro splicing-complementation system derived from mammalian cells to analyze in detail the sequence requirements of human 6 RNA during the first and second steps of splicing, focusing on the central domain, including the conserved ACAGAGA sequence, and the adjacent helix I region. Such an in vitro analysis provides the important advantage that each step of the spliceosome cycle can be investigated separately; in particular, a function in the first catalytic stage of the splicing reaction can be clearly distinguished from a splicing defect due to prior assembly deficiencies, in contrast to in vivo assays. Our mutational analysis provided no evidence for splicingessential positions upstream of the conserved ACAGAGA sequence: A series of triple substitutions between nt 20 and nt 40, which should disturb the proposed 6/pre-mRNA base pairing (23-25), revealed no dramatic phenotypes. This result is consistent with previous mutant studies in yeast (15) and Xenopus (27). Such a base-pairing interaction, however, may exist and contribute to the splicing efficiency of specific pre-mrnas but may not be essential for the general splicing mechanism. Evidence for two functionally important elements of 6 RNA, the ACAGAGA sequence and helix I, relied primarily on mutational analyses in yeast. On the basis of our present study, 6 RNA sequence requirements can now be directly compared between the yeast and mammalian systems. Within the conserved ACAGAGA box, position 5 (ACA- GAGA: human 6 A45 and yeast 6 A51) is important for the second step of the reaction in both systems. However, in contrast to yeast, the effect in the mammalian system is clearly base specific: and A45 -* C completely orpartially blocked the second step, respectively, but A45 -- G did not affect splicing complementation. An additional, relatively weak second-step defect at position 6 (ACAGAQA: human G46 and yeast G52) has been detected only in yeast. There are two differences of the first-step splicing effects observed: At position 2 (ACAGAGA, human C42 and yeast C4), we found no dramatic reduction in splicing activity, in contrast to yeast; at position 7 (ACAGAGA, human A47 and yeast A53) the A47 -- G and A47 -* point mutations reduced splicing activity to very low or intermediate levels, respectively, in the mammalian system, but no splicing effect had been detected in yeast. Apart from some minor quantitative differences, partial first-step splicing defects by mutations at the other positions were generally consistent between the two splicing systems. In the mammnalian system, we have demonstrated that the observed first-step effects result from defects occurring after spliceosome assembly. This result is in contrast to yeast, where 6 mutant RNAs with first-step splicing defects failed to assemble into spliceosomes (15). This apparent discrepancy might be reconciled by the assumption that analogous nucleotide

5 Biochemistry: Wofff et al. Proc. Natl. Acad. Sci. SA 91 (1994) 907 positions contribute differently to spliceosome stability in these systems. For example, a destabilizing effect may not become apparent in the mammalian system because the spliceosome can be held together through other interactions. Thus, analogous positions may, in fact, play similar roles during splicing in yeast and mammals. In sum, the ACA- GAGA box functions during both the first and second stage of mammalian pre-mrna splicing. The phylogenetic conservation of the 6-2 helix I argues for a particularly important function (ref. 8; see Fig. 5 for the human 6-2 helix I). A characteristic feature of the 6-2 helix I is its separation into two halves (helices Ia and Tb) by a 2-nt bulge in the 2 sequence. There are two minor differences between the yeast and the mammalian helix Ta-Ib junction: (i) the two bulged nucleotides are 24A25 in yeast and A23A24 in human 2 RNA; (ii) in yeast a G-C base pair terminates helix Ia (2 G26; 6 C58), corresponding to the human G- base pair (2 G ). How do the functional analyses of 6 mutations in helix I compare? In both the yeast and the mammalian system, base substitutions of the last 6 position in helix Ta (human 52 and yeast C58) resulted in a partial block of the second step, although with slightly different base specificity (15). Mutations leading to opposing guanosine residues in the 6-2 helix had the strongest effect, whereas G- and G-C base pairs were fully functional in both systems, consistent with the notion that base-pairing at this position, but not base identity, is critical for splicing function (11). An adenosine at this 6 position opposite the guanosine of 2 resulted in only a weak (yeast) or no detectable (human) second-step effect. Point mutations at the first position of helix Tb (human A53 and yeast A59) revealed somewhat different sequence requirements in the two splicing systems. In the mammalian in vitro system, both the A53 -) and A53 -* C mutations resulted in strong first-step effects; in contrast, in yeast second-step effects had been observed (15). The human A53 -* G mutation, which would convert the A into a G- 6-2 base pair, was tolerated, different from yeast where G- base-pairing at this position led to a second-step effect in vitro (15) and resulted in a lethal phenotype in vivo (11). We cannot exclude that position A53 has an additional, secondstep function also in the mammalian system; it might be obscured by the strong first-step defect of the A53 -, C mutation or by the block in 4-6 interaction of the A53 -+ mutation. For the two remaining, conserved positions of helix Tb (human G54C55 and yeast G6OC61), the effects of point mutations are qualitatively consistent between the two systems, demonstrating their importance for the first step of the splicing reaction. Significantly, the 6 and 2 positions at the helix Ta-Tb junction are critical for the second step of splicing (11, 15, 17; this study). We have observed a complete block of the second step by inserting 2 nt into 6, thus allowing a contiguous base-pairing through helices Ta and Tb. This result underlines the importance of the helix Ia-Ib arrangement specifically in the second step. In addition, we can conclude that neither helix Ta-Tb spacing and geometry nor their base-pairing stability is critical for the first reaction step. The conserved adenosine separating helix I from the intramolecular helix of 6 (yeast A62, human A56) appears not to be required for splicing, as point mutations (15) or a deletion (this study) showed. How can the discrepancies between mutational analyses in the yeast and mammalian system be explained? One possibility is that they may be due to different requirements of the various splicing substrates used; other possible explanations are related to differences between the yeast and mammalian in vitro systems; for example, limiting second-step splicing factors could facilitate the detection of weak effects on the second step. However, the critical 6 RNA positions identified in the mammalian system are similarly important in yeast; thus, the functional role of 6 in the splicing mechanism appears to be conserved. In an independent study, the effects of single-point mutations in the ACAGAGA sequence and in helix Ib on splicing in mammalian cells have been analyzed (28). The transient expression assay used did not allow distinction between first- and second-step splicing defects; surprisingly, a number ofpoint mutations with strong defects in yeast did not affect splicing activity in that mammalian in vivo system. Therefore, the mammalian splicing mechanism appeared more tolerant to 6 mutations than in yeast, and 6 was suggested to play a structural rather than a catalytic role, as considered by Vankan et al. (29). However, our data derived from a mammalian in vitro splicingcomplementation system are overall consistent with previous mutant analyses in yeast. Consequently, we find it more likely that the discrepancies reflect differences in the assay systems rather than intrinsic differences between the yeast and mammalian splicing mechanisms. In sum, our results have strengthened the arguments in favor of a catalytic role of 6 RNA in splicing. We acknowledge the excellent technical assistance of Heike Roscher, BjornWieland, and Manuela Staeber. We thank Mike Hearnefor oligonucleotide synthesis; Christine Guthrie, Alan Weiner, and Christiane Branlant for unpublished information; and Michael Cross for critical comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 344/C5). 1. Padgett, R. A., Grabowski, P. J., Konarska, M. M., Seiler, S. R. & Sharp, P. A. (1986) Annu. Rev. Biochem. 55, Steitz, J. A., Black, D. L., Gerke, V., Parker, K. A., Krimer, A., Frendewey, D. & Keller, W. (1988) in Structure and Function ofmajor and Minor Small NuclearRibonucleoprotein Particles, ed. Birnstiel, M. L. (Springer, Berlin), pp Green, M. R. (1991) Annu. Rev. Cell Biol. 7, Guthrie, C. (1991) Science 253, Sharp, P. (1985) Cell 42, Cech, T. R. (1986) Cell 44, Weiner, A. M. (1993) Cell 72, Guthrie, C. & Patterson, B. (1988) Annu. Rev. Genet. 22, Rinke, J., Appel, B., Digweed, M. & Luhrmann, R. (1985) J. Mol. Biol. 185, Brow, D. A. & Guthrie, C. (1988) Nature (London) 334, Madhani, H. D. & Guthrie, C. (1992) Cell 71, Wolff, T. & Bindereif, A. (1993) Genes Dev. 7, Datta, B. & Weiner, A. M. (1991) Nature (London) 352, Wu, J. & Manley, J. L. (1991) Nature (London) 352, Fabrizio, P. & Abelson, J. (1990) Science 250, Madhani, H. D., Bordonn6, R. & Guthrie, C. (1990) Genes Dev. 4, McPheeters, D. S. & Abelson, J. (1992) Cell 71, Wolff, T. & Bindereif, A. (1992) EMBO J. 11, Tani, T. & Oshima, Y. (1991) Genes Dev. 5, England, T. E., Bruce, A. G. & hlenbeck, 0. C. (1980) Methods Enzymol. 65, Zillmann, M., Zapp, M. L. & Berget, S. M. (1988) Mol. Cell. Biol. 8, Bindereif, A. & Green, M. R. (1987) EMBO J. 6, Sawa, H. & Abelson, J. (1992) Proc. Natl. Acad. Sci. SA 89, Sawa, H. & Shimura, Y. (1992) Genes Dev. 6, Wassarman, D. A. & Steitz, J. A. (1992) Science 257, Nelson, K. K. & Green, M. R. (1988) Genes Dev. 2, Vankan, P., McGuigan, C. & Mattaj, I. W. (1990) EMBO J. 9, Datta, B. & Weiner, A. M. (1993) Mol. Cell. Biol. 13, Vankan, P., McGuigan, C. & Mattaj, I. W. (1992) EMBO J. 11,