The EMBO Journal vol. 10 no. 5 pp. 1 209 - 1 21 6, 1 991
The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA Bertrand Seraphin' 2'3 and Michael Rosbash2 'Institut Curie-Biologie, Batiment 110, Campus Universitaire, 91405 Orsay Cedex, France and 2Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, MA 02254, USA 3Present address: EMBL, Meyerhofstrasse 1, D-6900 Heidelberg, FRG Communicated by P.A.Sharp
Commitment complexes contain Ul snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vit spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient Ul snRNA- pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a Ul snRNP- pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly. Key words: commitment complexes/intron recognition/ spliceosome/splice siteslSaccharomyces cerevisiae
Introduction Numerous analyses have shown that in vitro spliceosome assembly occurs in multiple steps which are likely to reflect the ordered addition of snRNPs and protein factors to the pre-mRNA (Frendewey and Keller, 1985; Konarska and Sharp, 1986, 1987; Pikielny et al., 1986; Bindereif and Green, 1987; Cheng and Abelson, 1987; Lamond et al., 1988; Blencowe et al., 1989). Ul snRNA was first suggested to be a splicing factor a decade ago (Lerner et al., 1980; Rogers and Wall, 1980). Although it was subsequently shown to be required for splicing (Kramer et al., 1984; Zhuang and Weiner, 1986), it was not observed associated with spliceosomes in these early studies. In higher eukaryotes, formation of U2 snRNP-containing complexes (or pre-spliceosomes) also seemed to occur in the absence of functional Ul snRNP (Frendewey et al., 1987; Kramer, 1987; Hamm et al., 1989). This was the case even in partially purified systems (Kramer, 1988; Zamore and Green, 1989). Pre-spliceosome formation also takes place on substrates lacking a 5' splice site (Frendewey and Keller, 1985; Konarska and Sharp, 1986; Bindereif and Green, 1987; Lamond et al., 1987). Because the 5' splice site had Oxford University Press
been shown to interact with Ul snRNA by base pairing (Zhuang and Weiner, 1986), these other observations reinforced the suggestion that Ul snRNP was not involved in U2 snRNP addition to the pre-mRNA. Very recent experiments indicate to the contrary, i.e. that Ul snRNP is involved in the formation of the U2 snRNP-pre-mRNA complex, although no interaction with the 5' splice site is required (Barabino et al., 1990). Another recent study reports the identification of a Ul snRNP-pre-mRNA complex during mammalian spliceosome formation (Reed, 1990). Further studies will be required to clarify the exact role of Ul snRNP in mammalian pre-spliceosome formation. In contrast, a relatively consistent picture of the early steps of spliceosome assembly has emerged from in vitro studies in yeast (Saccharomyces cerevisiae) extracts. Both the 5' splice site and branchpoint region of the pre-mRNA are required for pre-spliceosome formation (Pikielny and Rosbash, 1986; Cheng and Abelson, 1987; Rymond and Rosbash, 1988). The 5' splice site requirement is due in part to a requirement for a proper 5' splice site -Ul snRNA base pairing interaction before or concomitant with U2 snRNP binding (Seraphin et al., 1988). In the absence of active U2 snRNP, pre-mRNA is assembled into a functional stable complex, committed to the spliceosome and splicing pathway (a 'commitment complex'-Legrain et al., 1988). With the development of effective genetic procedures to prepare snRNP-depleted extracts, it was shown that commitment complex formation required U1 snRNP but took place in the absence of U2 snRNP (Seraphin and Rosbash, 1989). Further insight into commitment complex assembly was obtained with a novel native gel electrophoresis procedure. This technique revealed that commitment complexes could be fractionated into two sub-species, both of which contained Ul snRNP (Seraphin and Rosbash, 1989). Finally, indirect competition assays indicated that optimal commitment complex formation required two pre-mRNA cis-acting sequences, the 5' splice site (GUAUGU) and branchpoint region (UACUAAC) (Legrain et al., 1988; Seraphin and Rosbash, 1989). Similar conclusions were independently drawn by Ruby and Abelson (1988). Although the affinity chromatography procedure used in that study could not address the functional relevance of the observed pre-mRNA-containing complexes, U 1 snRNP binding seemed to precede and was necessary for subsequent U2 snRNP binding. Also, direct assays of mutant substrates showed that the formation of the U 1 snRNP-pre-mRNA complex required a proper branchpoint sequence as well as a proper 5' splice site. In this communication, we have used our native gel electrophoresis procedures and U2 snRNP-depleted extracts to analyze directly the role of pre-mRNA cis-acting sequences in the formation of functional Ul snRNP complexes, i.e. commitment complexes (Seraphin and Rosbash, 1989). Ul snRNP binding required a proper 5' splice site but did not require a branchpoint sequence.
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B.S6raphin and M.Rosbash
However, one of the two commitment complex sub-species was absolutely dependent on a wild-type branchpoint sequence, consistent with the notion that this cis-acting sequence is recognized prior to U2 snRNP binding during spliceosome assembly.
binding. With all three branchpoint mutants, large amounts of commitment complex formation took place (lanes 4-6), but only a single band that comigrated with CC1 was detected. We will refer to this complex also as CC1 although we do not know that this complex is identical to the CC 1 complex formed with the wild-type substrates. Complex formation with the branchpoint mutant substrates occurred in the absence of added ATP but required Ul snRNP (data not shown, see also Legrain et al., 1988; Seraphin and Rosbash, 1989). In a second set of experiments, complex formation was assayed after incubating the same substrates in a complete whole cell extract (Figure 2B). Spliceosome formation was observed with both the WT-A2 and WT-B substrates (lanes 1 and 2, respectively), whereas only traces of splicing complexes were detected when the 5' splice site GUAUaU mutant substrate was used (lane 3) as expected from previous observations (Seraphin et al., 1988). With the three branchpoint mutant substrates, only a single complex comigrating with CC 1 was observed (lanes 3-5). As these results were indistinguishable from those obtained with the branchpoint mutant substrates in the U2-depleted extract (Figure 2A), we conclude that they were not due to the depletion procedure but that these mutant substrates were unable to proceed past this early stage of the spliceosome assembly pathway. To examine further the role of the 3' splice site region in commitment complex formation, we generated truncated pre-mRNA substrates by cleaving the DNA templates with the restriction enzyme Nsil before in vitro transcription (see Figure 1). Control transcripts were synthesized after cleavage of the templates at the DdeI site 32 nucleotides downstream of the 3' splice site. After incubation in extract, the complexes were resolved by native gel electrophoresis (Figure 2C). The WT-B substrate formed spliceosomes in a complete extract and commitment complexes in a U2 snRNP-depleted extract, with or without a 3' splice site (compare lane 1 with lane 2, and lane 3 with lane 7, respectively; see also Rymond and Rosbash, 1985; Rymond et al., 1987). Similarly, the
Results Construction of mutant pre-mRNA substrates To study the effects of intron cis-acting sequences on commitment complex formation, we constructed a set of mutant pre-mRNA substrates. The starting plasmid was a T7 promoter-containing vector into which the WT-A2 intron had been inserted (see Materials and methods). Because the WT-A2 intron contains a pseudo-branchpoint sequence (UACaAAC, Figure 1) that might complicate interpretation, this region was mutagenized and changed into an NsiI restriction site. The resulting construct was named WT-B. A 5' splice site mutation (GUAUaU, formerly called 5'II, Jacquier et al., 1985) and three mutations in the branchpoint region (UACaAAC; AUACUAAC and A2-3B) were then independently introduced into the WT-B background (Figure 1).
Complex formation using 5' splice site and branchpoint mutant substrates Full length RNA substrates were incubated in a U2 snRNPdepleted (U2-depleted) extract and the complexes resolved by native gel electrophoresis (Figure 2A). The two Ul snRNP-containing commitment complex bands previously described (Seraphin and Rosbash, 1989) were observed with both the WT-A2 and the WT-B substrates (lanes 1 and 2). Interestingly, the ratio of the two bands (i.e. the amount of faster mobility complex, CCl, compared with the amount of slower mobility complex, CC2) differed slightly between the two 'wild-type' substrates. This is consistent with some interference by the pseudo-branchpoint sequence of the WT-A2 construct with formation of the more mature CC2 complex (see below). With the 5' splice site mutant substrate, only trace amounts of complex were observed (lane 3), consistent with the idea that a proper 5' splice site is important for stable Ul snRNP 5'splice site
-A2 WT-A2
Branch point
Pseudo branch
~ ____point
3 'splice sit
site
-AAUG GUAUGUUAAUA--56nt--UCAGUAUACUAACAAGUUGAAUUGCAUUUACAAACUUUU--23nt--UUUAAUAG GGU-
NsiI
WT-B
-AAUG
GUAUAU
-AAUG GUAUaUUAAUA--5bnt--UCAGUAUACUAACAAGUUGAAUUGCAUUCAUGCAUWUUU-- -23nt--UUUAAUAG
UACAAAC
-AAUG GUAUGUUAAUA--5 6nt--UCAGUAUACeaACAAGUUGAAUUGCAUUCAUGCAUUUUU--2 3nt--UUUAAUAG GGU-
AUACUAAC
-AAUG GUAUGUUAAUA--5 6nt--UCAGUA ....
A2-3B
-AAUG
GGUGUAGGUUAAUA--56nt--UCAGUAUACUAACAAGUUGAAUUGCAUUCAUGCAUUUUU--23nt--UUUAAUAG
GGU-
AA...GUUGAAUUGCAUUCAUGCAUWUUU--2 3nt--UUUAAUAG
GGU-
GUAUGUUAAUA--56nt--UCAGUAU ............................. UUU--23nt--UUUAAUAG
GGU-
Fig. 1. Primary sequences of the various substrates. Sequences encompassing the 5' splice site, branchpoint region and 3' splice site of the RP5lA derivatives used in this study are depicted. The names of constructs are shown preceding the corresponding sequences. The pseudo-branchpoint sequence present in the WT-A2 construct is indicated; in the other constructs this sequence has been replaced by an NsiI restriction site. Dots indicate deleted nucleotides.
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Branchpoint sequence role in spliceosome assembly
branchpoint deletion mutant substrate formed CC l with or without a 3' splice site (lanes 5 and 9). These results indicate that CCl formation can occur in the absence of both a branchpoint sequence and a 3' splice site, suggesting that the only highly conserved pre-mRNA cis-acting sequence required for formation of a U1 snRNP-containing complex is the 5' splice site. As expected, little or no specific complexes were detectable with the full length or truncated 5' splice site GUAUaU mutant substrate (lanes 4 and 8) or with non-specific substrates of similar lengths (lanes 6 and 10). In summary, a 5' splice site mutant blocked commitment complex formation, whereas deletion of the 3' splice site did not affect this process. An intermediate situation obtains for the branchpoint sequence, as it was not required for formation of the CCl complex but was required for CC2 formation.
Commitment complex formation using actin substrates The observations described above were consistent with our previous competition experiments that indicated a role for the branchpoint as well as the 5' splice site prior to U2 snRNP addition (e.g. Seraphin and Rosbash, 1989). The branchpoint independence of commitment complex formation (CC1 formation) was surprising, however, because Ruby and Abelson (1988) had reported that Ul snRNP binding to the pre-mRNA requires a wild-type UACUAAC sequence. Because actin gene-derived substrates were used in that study, we analyzed commitment complex formation using the same substrates (Figure 3). The wild-type actin substrate formed spliceosomes in a complete extract (lane 6) and the two expected commitment complex bands in a U2-depleted extract (lane 2). In both a U2-depleted extract and a complete extract, the actin branchpoint mutant (A257,
C -o.Ds:ra:&
A
.'
The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA Bertrand Seraphin' 2'3 and Michael Rosbash2 'Institut Curie-Biologie, Batiment 110, Campus Universitaire, 91405 Orsay Cedex, France and 2Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, MA 02254, USA 3Present address: EMBL, Meyerhofstrasse 1, D-6900 Heidelberg, FRG Communicated by P.A.Sharp
Commitment complexes contain Ul snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vit spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient Ul snRNA- pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a Ul snRNP- pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly. Key words: commitment complexes/intron recognition/ spliceosome/splice siteslSaccharomyces cerevisiae
Introduction Numerous analyses have shown that in vitro spliceosome assembly occurs in multiple steps which are likely to reflect the ordered addition of snRNPs and protein factors to the pre-mRNA (Frendewey and Keller, 1985; Konarska and Sharp, 1986, 1987; Pikielny et al., 1986; Bindereif and Green, 1987; Cheng and Abelson, 1987; Lamond et al., 1988; Blencowe et al., 1989). Ul snRNA was first suggested to be a splicing factor a decade ago (Lerner et al., 1980; Rogers and Wall, 1980). Although it was subsequently shown to be required for splicing (Kramer et al., 1984; Zhuang and Weiner, 1986), it was not observed associated with spliceosomes in these early studies. In higher eukaryotes, formation of U2 snRNP-containing complexes (or pre-spliceosomes) also seemed to occur in the absence of functional Ul snRNP (Frendewey et al., 1987; Kramer, 1987; Hamm et al., 1989). This was the case even in partially purified systems (Kramer, 1988; Zamore and Green, 1989). Pre-spliceosome formation also takes place on substrates lacking a 5' splice site (Frendewey and Keller, 1985; Konarska and Sharp, 1986; Bindereif and Green, 1987; Lamond et al., 1987). Because the 5' splice site had Oxford University Press
been shown to interact with Ul snRNA by base pairing (Zhuang and Weiner, 1986), these other observations reinforced the suggestion that Ul snRNP was not involved in U2 snRNP addition to the pre-mRNA. Very recent experiments indicate to the contrary, i.e. that Ul snRNP is involved in the formation of the U2 snRNP-pre-mRNA complex, although no interaction with the 5' splice site is required (Barabino et al., 1990). Another recent study reports the identification of a Ul snRNP-pre-mRNA complex during mammalian spliceosome formation (Reed, 1990). Further studies will be required to clarify the exact role of Ul snRNP in mammalian pre-spliceosome formation. In contrast, a relatively consistent picture of the early steps of spliceosome assembly has emerged from in vitro studies in yeast (Saccharomyces cerevisiae) extracts. Both the 5' splice site and branchpoint region of the pre-mRNA are required for pre-spliceosome formation (Pikielny and Rosbash, 1986; Cheng and Abelson, 1987; Rymond and Rosbash, 1988). The 5' splice site requirement is due in part to a requirement for a proper 5' splice site -Ul snRNA base pairing interaction before or concomitant with U2 snRNP binding (Seraphin et al., 1988). In the absence of active U2 snRNP, pre-mRNA is assembled into a functional stable complex, committed to the spliceosome and splicing pathway (a 'commitment complex'-Legrain et al., 1988). With the development of effective genetic procedures to prepare snRNP-depleted extracts, it was shown that commitment complex formation required U1 snRNP but took place in the absence of U2 snRNP (Seraphin and Rosbash, 1989). Further insight into commitment complex assembly was obtained with a novel native gel electrophoresis procedure. This technique revealed that commitment complexes could be fractionated into two sub-species, both of which contained Ul snRNP (Seraphin and Rosbash, 1989). Finally, indirect competition assays indicated that optimal commitment complex formation required two pre-mRNA cis-acting sequences, the 5' splice site (GUAUGU) and branchpoint region (UACUAAC) (Legrain et al., 1988; Seraphin and Rosbash, 1989). Similar conclusions were independently drawn by Ruby and Abelson (1988). Although the affinity chromatography procedure used in that study could not address the functional relevance of the observed pre-mRNA-containing complexes, U 1 snRNP binding seemed to precede and was necessary for subsequent U2 snRNP binding. Also, direct assays of mutant substrates showed that the formation of the U 1 snRNP-pre-mRNA complex required a proper branchpoint sequence as well as a proper 5' splice site. In this communication, we have used our native gel electrophoresis procedures and U2 snRNP-depleted extracts to analyze directly the role of pre-mRNA cis-acting sequences in the formation of functional Ul snRNP complexes, i.e. commitment complexes (Seraphin and Rosbash, 1989). Ul snRNP binding required a proper 5' splice site but did not require a branchpoint sequence.
1209
B.S6raphin and M.Rosbash
However, one of the two commitment complex sub-species was absolutely dependent on a wild-type branchpoint sequence, consistent with the notion that this cis-acting sequence is recognized prior to U2 snRNP binding during spliceosome assembly.
binding. With all three branchpoint mutants, large amounts of commitment complex formation took place (lanes 4-6), but only a single band that comigrated with CC1 was detected. We will refer to this complex also as CC1 although we do not know that this complex is identical to the CC 1 complex formed with the wild-type substrates. Complex formation with the branchpoint mutant substrates occurred in the absence of added ATP but required Ul snRNP (data not shown, see also Legrain et al., 1988; Seraphin and Rosbash, 1989). In a second set of experiments, complex formation was assayed after incubating the same substrates in a complete whole cell extract (Figure 2B). Spliceosome formation was observed with both the WT-A2 and WT-B substrates (lanes 1 and 2, respectively), whereas only traces of splicing complexes were detected when the 5' splice site GUAUaU mutant substrate was used (lane 3) as expected from previous observations (Seraphin et al., 1988). With the three branchpoint mutant substrates, only a single complex comigrating with CC 1 was observed (lanes 3-5). As these results were indistinguishable from those obtained with the branchpoint mutant substrates in the U2-depleted extract (Figure 2A), we conclude that they were not due to the depletion procedure but that these mutant substrates were unable to proceed past this early stage of the spliceosome assembly pathway. To examine further the role of the 3' splice site region in commitment complex formation, we generated truncated pre-mRNA substrates by cleaving the DNA templates with the restriction enzyme Nsil before in vitro transcription (see Figure 1). Control transcripts were synthesized after cleavage of the templates at the DdeI site 32 nucleotides downstream of the 3' splice site. After incubation in extract, the complexes were resolved by native gel electrophoresis (Figure 2C). The WT-B substrate formed spliceosomes in a complete extract and commitment complexes in a U2 snRNP-depleted extract, with or without a 3' splice site (compare lane 1 with lane 2, and lane 3 with lane 7, respectively; see also Rymond and Rosbash, 1985; Rymond et al., 1987). Similarly, the
Results Construction of mutant pre-mRNA substrates To study the effects of intron cis-acting sequences on commitment complex formation, we constructed a set of mutant pre-mRNA substrates. The starting plasmid was a T7 promoter-containing vector into which the WT-A2 intron had been inserted (see Materials and methods). Because the WT-A2 intron contains a pseudo-branchpoint sequence (UACaAAC, Figure 1) that might complicate interpretation, this region was mutagenized and changed into an NsiI restriction site. The resulting construct was named WT-B. A 5' splice site mutation (GUAUaU, formerly called 5'II, Jacquier et al., 1985) and three mutations in the branchpoint region (UACaAAC; AUACUAAC and A2-3B) were then independently introduced into the WT-B background (Figure 1).
Complex formation using 5' splice site and branchpoint mutant substrates Full length RNA substrates were incubated in a U2 snRNPdepleted (U2-depleted) extract and the complexes resolved by native gel electrophoresis (Figure 2A). The two Ul snRNP-containing commitment complex bands previously described (Seraphin and Rosbash, 1989) were observed with both the WT-A2 and the WT-B substrates (lanes 1 and 2). Interestingly, the ratio of the two bands (i.e. the amount of faster mobility complex, CCl, compared with the amount of slower mobility complex, CC2) differed slightly between the two 'wild-type' substrates. This is consistent with some interference by the pseudo-branchpoint sequence of the WT-A2 construct with formation of the more mature CC2 complex (see below). With the 5' splice site mutant substrate, only trace amounts of complex were observed (lane 3), consistent with the idea that a proper 5' splice site is important for stable Ul snRNP 5'splice site
-A2 WT-A2
Branch point
Pseudo branch
~ ____point
3 'splice sit
site
-AAUG GUAUGUUAAUA--56nt--UCAGUAUACUAACAAGUUGAAUUGCAUUUACAAACUUUU--23nt--UUUAAUAG GGU-
NsiI
WT-B
-AAUG
GUAUAU
-AAUG GUAUaUUAAUA--5bnt--UCAGUAUACUAACAAGUUGAAUUGCAUUCAUGCAUWUUU-- -23nt--UUUAAUAG
UACAAAC
-AAUG GUAUGUUAAUA--5 6nt--UCAGUAUACeaACAAGUUGAAUUGCAUUCAUGCAUUUUU--2 3nt--UUUAAUAG GGU-
AUACUAAC
-AAUG GUAUGUUAAUA--5 6nt--UCAGUA ....
A2-3B
-AAUG
GGUGUAGGUUAAUA--56nt--UCAGUAUACUAACAAGUUGAAUUGCAUUCAUGCAUUUUU--23nt--UUUAAUAG
GGU-
AA...GUUGAAUUGCAUUCAUGCAUWUUU--2 3nt--UUUAAUAG
GGU-
GUAUGUUAAUA--56nt--UCAGUAU ............................. UUU--23nt--UUUAAUAG
GGU-
Fig. 1. Primary sequences of the various substrates. Sequences encompassing the 5' splice site, branchpoint region and 3' splice site of the RP5lA derivatives used in this study are depicted. The names of constructs are shown preceding the corresponding sequences. The pseudo-branchpoint sequence present in the WT-A2 construct is indicated; in the other constructs this sequence has been replaced by an NsiI restriction site. Dots indicate deleted nucleotides.
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Branchpoint sequence role in spliceosome assembly
branchpoint deletion mutant substrate formed CC l with or without a 3' splice site (lanes 5 and 9). These results indicate that CCl formation can occur in the absence of both a branchpoint sequence and a 3' splice site, suggesting that the only highly conserved pre-mRNA cis-acting sequence required for formation of a U1 snRNP-containing complex is the 5' splice site. As expected, little or no specific complexes were detectable with the full length or truncated 5' splice site GUAUaU mutant substrate (lanes 4 and 8) or with non-specific substrates of similar lengths (lanes 6 and 10). In summary, a 5' splice site mutant blocked commitment complex formation, whereas deletion of the 3' splice site did not affect this process. An intermediate situation obtains for the branchpoint sequence, as it was not required for formation of the CCl complex but was required for CC2 formation.
Commitment complex formation using actin substrates The observations described above were consistent with our previous competition experiments that indicated a role for the branchpoint as well as the 5' splice site prior to U2 snRNP addition (e.g. Seraphin and Rosbash, 1989). The branchpoint independence of commitment complex formation (CC1 formation) was surprising, however, because Ruby and Abelson (1988) had reported that Ul snRNP binding to the pre-mRNA requires a wild-type UACUAAC sequence. Because actin gene-derived substrates were used in that study, we analyzed commitment complex formation using the same substrates (Figure 3). The wild-type actin substrate formed spliceosomes in a complete extract (lane 6) and the two expected commitment complex bands in a U2-depleted extract (lane 2). In both a U2-depleted extract and a complete extract, the actin branchpoint mutant (A257,
C -o.Ds:ra:&
A
.'
