Unconventional Splicing of HAC1/ERN4 mRNA Required for the Unfolded Protein Response

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signaling pathway from the ER to the nucleus, termed the unfolded protein response. We and others recently identified transcription factor Hac1p/Ern4p responsible for the response in Saccharomyces cerevisiaeand found that Hac1p expression is controlled by the regulated splicing of HAC1 mRNA. Walter and co-workers (Sidrauski, C., Cox, J. S., and Walter, P. (1996) Cell, 87, 405–413) further showed that the splicing requires tRNA ligase but not spliceosome. In this report, we carried out mutational analysis ofHAC1 mRNA and revealed several unique features of the splicing. First, a mutation or deletion of the branchpoint-like sequence present in HAC1 intron did not affect the splicing. Second, cleavage of the splice sites was sequence-specific and thus completely blocked by some point mutations introduced at the 5′ or 3′ splice site. Third, cleavage of the 5′ and 3′ splice sites could occur independently as judged by the nature of splicing intermediates accumulated. Fourth, swapping the nucleotide sequences of the 5′ and 3′ splice sites inhibited the ligation but not the cleavage step. We conclude that signaling from the ER activates putative endonucleases that can carry out sequence-specific cleavage of the splice sites in a random order. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signaling pathway from the ER to the nucleus, termed the unfolded protein response. We and others recently identified transcription factor Hac1p/Ern4p responsible for the response in Saccharomyces cerevisiaeand found that Hac1p expression is controlled by the regulated splicing of HAC1 mRNA. Walter and co-workers (Sidrauski, C., Cox, J. S., and Walter, P. (1996) Cell, 87, 405–413) further showed that the splicing requires tRNA ligase but not spliceosome. In this report, we carried out mutational analysis ofHAC1 mRNA and revealed several unique features of the splicing. First, a mutation or deletion of the branchpoint-like sequence present in HAC1 intron did not affect the splicing. Second, cleavage of the splice sites was sequence-specific and thus completely blocked by some point mutations introduced at the 5′ or 3′ splice site. Third, cleavage of the 5′ and 3′ splice sites could occur independently as judged by the nature of splicing intermediates accumulated. Fourth, swapping the nucleotide sequences of the 5′ and 3′ splice sites inhibited the ligation but not the cleavage step. We conclude that signaling from the ER activates putative endonucleases that can carry out sequence-specific cleavage of the splice sites in a random order. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Bp, branchpoint; PCR, polymerase chain reaction; pre-mRNA, precursor mRNA; UPR, unfolded protein-response; UPRE, unfolded protein-response element; nt, nucleotide(s); kb, kilobase(s); aa, amino acid(s); WT, wild-type. activates an intracellular signaling pathway from the ER to the nucleus, resulting in transcriptional induction of molecular chaperones and folding enzymes localized in the ER (1Lee A.S. Trends Biochem. Sci. 1987; 12: 20-23Abstract Full Text PDF Scopus (394) Google Scholar, 2Kozutsumi Y. Segal M. Normington K. Gething M.J. Sambrook J. Nature. 1988; 332: 462-464Crossref PubMed Scopus (996) Google Scholar, 3McMillan D.R. Gething M.J. Sambrook J. Curr. Opin. Biotech. 1994; 5: 540-545Crossref PubMed Scopus (73) Google Scholar, 4Shamu C.E. Cox J.S. Walter P. Trends Cell Biol. 1994; 4: 56-60Abstract Full Text PDF PubMed Scopus (133) Google Scholar). This induction system, termed the unfolded protein response (UPR), is observed in all eukaryotes examined and is required for survival under the conditions that continuously accumulate unfolded proteins in the ER (“ER stress”) in budding yeast Saccharomyces cerevisiae (5Cox J.S. Shamu C.E. Walter P. Cell. 1993; 73: 1197-1206Abstract Full Text PDF PubMed Scopus (950) Google Scholar, 6Mori K. Ma W. Gething M.J. Sambrook J. Cell. 1993; 74: 743-756Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 7Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (306) Google Scholar, 8Nikawa J. Akiyoshi M. Hirata S. Fukuda T. Nucleic Acids Res. 1996; 24: 4222-4226Crossref PubMed Scopus (85) Google Scholar) as well as in mammalian cells (9Li X. Lee A.S. Mol. Cell. Biol. 1991; 11: 3446-3453Crossref PubMed Scopus (69) Google Scholar, 10Li L.J. Li X. Ferrario A. Rucker N. Liu E.S. Wong S. Gomer C.J. Lee A.S. J. Cell. Physiol. 1992; 153: 575-582Crossref PubMed Scopus (92) Google Scholar, 11Little E. Lee A.S. J. Biol. Chem. 1995; 270: 9526-9534Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Morris J.A. Dorner A.J. Edwards C.A. Hendershot L.M. Kaufman R.J. J. Biol. Chem. 1997; 272: 4327-4334Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar), suggesting that the UPR has been quite important and conserved during evolution of eukaryotic cells. We and others recently demonstrated that a basic leucine zipper protein Hac1p/Ern4p functions as the transcription factor responsible for the UPR in S. cerevisiae; haploid cells lacking Hac1p (hac1Δ) are unable to induce transcription of any of the target genes of the UPR and exhibit sensitivity to ER stress (7Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (306) Google Scholar, 8Nikawa J. Akiyoshi M. Hirata S. Fukuda T. Nucleic Acids Res. 1996; 24: 4222-4226Crossref PubMed Scopus (85) Google Scholar,13Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). Furthermore, Hac1p expression was found to be regulated posttranscriptionally in a completely unexpected manner;HAC1 mRNA is constitutively expressed but becomes spliced in response to ER stress (13Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). Thus, an intron of 252 nt is removed from 1.4-kb precursor mRNA (pre-mRNA) to produce 1.2-kb mature mRNA (see Fig. 1 A). The splicing event entirely depends on the signaling from the ER, and expression of mature mRNA activates the UPR. Since the 5′ splice site is located within the coding region, this splicing replaces the C-terminal portion of Hac1p. Pre- and mature mRNAs encode a protein of 230 and 238 aa, respectively, although these two types of Hac1p are supposed to share identical N-terminal 220 aa. Interestingly, only ER-stressed cells produce detectable amounts of Hac1p of 238 aa, which is thus translated from mature mRNA. Although Cox and Walter (13Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar) ascribed the absence of 230-aa-Hac1p potentially synthesized from pre-mRNA to its extreme instability, we showed that there is essentially no difference in stability between 230-aa- and 238-aa-Hac1p and that the absence of 230-aa-Hac1p is due to the lack of translation of pre-mRNA. Namely, Hac1p is synthesized only after the mRNA splicing takes place, leading to activation of the UPR (14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). This splicing is also quite unique in that sequences around the 5′ and 3′ splice sites do not match the consensus found in S. cerevisiae and higher eukaryotes (GT-AG or AT-AC; Refs. 15Kreivi J.-P. Lamond A.I. Curr. Biol. 1996; 6: 802-805Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar and16Tarn W.-Y. Steitz J.A. Trends Biochem. Sci. 1997; 22: 132-137Abstract Full Text PDF PubMed Scopus (170) Google Scholar). Walter and co-workers (17Sidrauski C. Cox J.S. Walter P. Cell. 1996; 87: 405-413Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) further showed that splicing ofHAC1 pre-mRNA is not mediated by the conventional pre-mRNA processing system. The splicing was not affected by conditional mutation of two components of the spliceosome (prp2 ts and prp8 ts), and tRNA ligase was found to be directly involved in the final step of the splicing, joining the two exons after ER stress-induced cleavage ofHAC1 pre-mRNA. In this report, we took a different approach to gain insight into the mechanism of this unconventional type of mRNA splicing. The results obtained by mutational analysis ofHAC1 mRNA will be discussed in relation to the features known for conventional pre-mRNA splicing as well as tRNA splicing. The yeast strain used in this study was KMY1145 (MATα leu2–3, 112 ura3–52 his3-Δ200 trp1-Δ901 lys2–801 hac1Δ::TRP1 ura3–52::URA3-UPRE-CYC1-lacZ) (14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). The composition of synthetic complete medium used for selection of transformants such as SC(-Ura, Leu) has been described (18Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). Tunicamycin was obtained from Sigma (T-7765) and used at a concentration of 5 μg/ml throughout the experiments. Transformation of yeast cells was performed by the lithium acetate method (19Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Recombinant DNA techniques were carried out as described (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Second Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The parental single-copy plasmids carrying the HAC1 gene, YCp-HAC1WT, and YCp-HAC1WT(XbaI), were described previously (14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). Plasmids with some mutated HAC1, YCp-HAC1-mBp and YCp-HAC1-ΔSL3 (Fig. 1), were constructed by site-directed mutagenesis (21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). Other mutations were introduced into YCp-HAC1WT(XbaI) by replacing the 0.18-kb XbaI640-HindIII818 or 0.11-kbHindIII818-EcoRI930 fragment with the corresponding fragment of the product obtained by polymerase chain reaction (PCR)-mediated mutagenesis after its sequence had been confirmed. Northern blot hybridization analysis was carried out as described previously (6Mori K. Ma W. Gething M.J. Sambrook J. Cell. 1993; 74: 743-756Abstract Full Text PDF PubMed Scopus (655) Google Scholar, 7Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (306) Google Scholar,14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). The positions of probes specific for the first or second exon ofHAC1 are illustrated in Fig. 1 A. Cellular UPR activity was monitored by measuring the level of β-galactosidase expressed from the UPRE-CYC1-lacZ reporter gene that had been integrated into the chromosome of KMY1145. Induction of β-galactosidase by tunicamycin entirely depends on both the splicing of HAC1pre-mRNA induced by the signaling from the ER and the direct interaction between Hac1p thus produced and cis-acting unfolded protein-response element (UPRE) (7Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (306) Google Scholar, 14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). Assays for β-galactosidase activity in yeast were carried out as described previously (6Mori K. Ma W. Gething M.J. Sambrook J. Cell. 1993; 74: 743-756Abstract Full Text PDF PubMed Scopus (655) Google Scholar). We utilized the mfold server by Zuker and Turner on the internet 2http://www.ibc.wustl.edu/~zuker/rna. to obtain a possible secondary structure of HAC1pre-mRNA at 30 °C. All of the nine structures obtained with minimum energy lower than −421.6 kcal/mol gave rise to an identical secondary structure for the intron-containing region which, as shown in Fig. 1 B, contained four stem-loop structures (designated SL1 to SL4 from the 5′ side). Interestingly, the 5′ or 3′ splice site was predicted to be localized in the loop of SL1 or SL4, respectively, each loop consisting of seven nucleotides (Fig. 2). A branchpoint (Bp)-like sequence (UACUAAG) present in HAC1intron (17Sidrauski C. Cox J.S. Walter P. Cell. 1996; 87: 405-413Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) was found around the loop of SL3. The Bp sequence known to be almost invariant in S. cerevisiae (UACUAAC) is utilized to form the lariat during the first step in conventional pre-mRNA splicing and is also important for of splicing as well as J.A. K. N. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar and The sequence in HAC1 intron is found nucleotides of the 3′ splice which well with Bp sequences located nucleotides of the 3′ splice sites J.-P. Lamond A.I. Curr. Biol. 1996; 6: 802-805Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We thus mutated the sequence and examined its on splicing of HAC1 mRNA induced by which is known to ER stress by of synthesized leading to activation of the UPR Y. Segal M. Normington K. Gething M.J. Sambrook J. Nature. 1988; 332: 462-464Crossref PubMed Scopus (996) Google Scholar, Trends Biochem. Sci. 6: Full Text PDF Scopus Google Scholar). In the strain carrying the on a single-copy expression to in Fig. 1 as well as splicing of constitutively expressed 1.4-kb HAC1pre-mRNA was induced within 1 after of tunicamycin to produce 1.2-kb mature mRNA as previously (13Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). these β-galactosidase expressed from the UPRE-CYC1-lacZ reporter gene was induced (Fig. 1 the sequence intron was mutated from to the and the resulting HAC1 gene was introduced into the the splicing and β-galactosidase was well We further the from HAC1 (designated the deletion did not affect the splicing ofHAC1 pre-mRNA or induction of β-galactosidase results that the sequence in HAC1 intron is not required for the regulated splicing of HAC1 pre-mRNA. we examined nucleotide sequences around the splice sites were by putative endonucleases during the splicing point mutations at the 5′ or 3′ splice we carried out mutagenesis a ofHAC1 YCp-HAC1WT(XbaI) as described under Although YCp-HAC1WT(XbaI) contained an site at nucleotide that to the two amino did not affect the level of mRNA or Hac1p but only the transcriptional activity of Hac1p (14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). A point mutation was introduced into each of the seven nucleotides predicted to form a loop structure of SL1 splice or SL4 splice and nucleotide each at both was to A was to and that the was not (Fig. 2). nucleotides at the 5′ or 3′ of the cleavage site were or of each point into the strain and after of in the or absence of β-galactosidase activity expressed from the reporter gene was and the of induction was with that of the (Fig. and of the point mutations were found to the others were Interestingly, four out of the nine nucleotides and in Fig. conserved between the 5′ and 3′ splice sites were for the a point mutation of any of these nucleotides induction of β-galactosidase almost with that affected the nucleotide of the 3′ splice site. the the nucleotide at of splice site not to be and the of point mutations at and between the 5′ and 3′ splice results that sequence of the splice sites is important for the UPR and that putative endonucleases nucleotide sequences at both of the splice Northern blot hybridization analysis was carried out to which step of the splicing was blocked by point mutations introduced at the 5′ or 3′ splice site from transformants for 1 in the or absence of tunicamycin were a specific for the first exon or the second exon of In cells carrying YCp-HAC1WT(XbaI), constitutively expressed 1.4-kb pre-mRNA 1 and was spliced by tunicamycin to produce 1.2-kb mature mRNA and In this a of was with specific for the first exon as with the by an in Fig. 1 this was not specific for the second exon (Fig. or intron not only of the first a of with specific for the second exon only of the second was higher than that obtained with not the amounts of these intermediates are to from ligation of the two exons due to the nucleotide introduced to site at nucleotide nucleotides of the 5′ splice site. the introduced affected The amounts of mature mRNA in cells carrying of HAC1 (Fig. were well with UPR by β-galactosidase (Fig. 2). no mature mRNA was a point mutation was introduced at of the 5′ splice site (Fig. or at of the 3′ splice site were under these In cells carrying a point mutation at of the 5′ splice specific for the first exon a of that was not with for the second exon but was with specific for intron not Thus, the a splicing consisting of the first exon and In the consisting of only the first exon was but the consisting of only the second exon was results that the point mutation at of the 5′ splice site completely blocked the cleavage at the 5′ splice site the cleavage at the 3′ splice site. almost identical was obtained a point mutation was introduced at of the 5′ splice site not the a point mutation at of the 5′ splice site showed on the splicing than the was induced (Fig. and a of mature mRNA was (Fig. In this two intermediates were which were to of the first exon and intron or only the first exon by hybridization to the probes that the cleavage at the 5′ splice site was but not blocked completely by the mutations at and of the 5′ splice site produced not In in cells carrying a point mutation at of the 3′ splice two at around the fragment were with specific for the second exon or with specific for intron not but not with for the first exon with a of probes specific to 5′ or 3′ of the intron that the the 5′ of intron not Thus, these splicing intermediates consisting of the second exon and intron of different Furthermore, the consisting of only the second exon was and level of the consisting of only the first exon was results that the point mutation at of the 3′ splice site completely blocked the cleavage at the 3′ splice site the cleavage at the 5′ splice site. mutations at of the 3′ splice site showed almost identical not these we that at the 5′ or 3′ splice site can occur not only independently from each but in a random order. we of HAC1 by swapping the nucleotides around the 5′ splice site and around the 3′ splice and examined on the UPR (Fig. shown in Fig. not only seven nucleotides predicted to be localized in the loop but also two nucleotides at the of and to be important for the the to mutation at of the 5′ splice site completely blocked the cleavage at the 5′ splice the mutation at of the 3′ splice site affected the splicing only we a of nine nucleotides to these are of the of two different endonucleases in two splice sites or contained nine nucleotides at the 5′ splice site with at the 3′ splice site contained nine nucleotides at the 3′ splice site with at the 5′ splice site In the nine nucleotides at the splice sites were entirely induction of β-galactosidase from the reporter gene was (Fig. and and activity of respectively, that the of the nucleotides at the 3′ splice site by at the 5′ splice site showed on the splicing. amounts of mature mRNA were by Northern blot hybridization in cells carrying each of the as with carrying the (Fig. of showed that each mature mRNA produced contained an predicted from the sequences of the splice sites not to the results shown in Fig. the splicing consisting of the first exon and intron or the second exon and intron was not with specific for the first or the second respectively, in cells carrying or and although the of induction was than of in these cells. amounts of the splicing consisting of only the first exon kb, or only the second exon kb, were that swapping the nine nucleotides inhibited the ligation but not the cleavage the that the cleavage at the 5′ and 3′ splice sites is by the In to of and two types of splicing, pre-mRNA splicing and tRNA splicing, and which revealed completely different In conventional pre-mRNA splicing, sequences at the 5′ and 3′ splice sites are conserved (GT-AG or and sequence-specific cleavage at the splice sites is by of and proteins in a spliceosome. In cleavage in two First, cleavage of the 5′ splice site in with the of between the of the intron and the in the Bp sequence located of the 3′ splice thus a lariat Second, cleavage of the 3′ splice site leading to of the lariat intron and ligation of the two exons J.-P. Lamond A.I. Curr. Biol. 1996; 6: 802-805Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, W.-Y. Steitz J.A. Trends Biochem. Sci. 1997; 22: 132-137Abstract Full Text PDF PubMed Scopus (170) Google Scholar, Curr. Opin. Cell Biol. 1997; PubMed Scopus Google Scholar and the tRNA splicing is by the of protein a a tRNA and a In to conventional pre-mRNA splicing, nucleotide sequences at the splice sites in are not and the cleavage at the two splice sites is by a in which a to the splice sites is from a in the mature of the Furthermore, the two splice sites are independently M. A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, F. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar and in two that the regulated splicing of mRNA transcription factor Hac1p/Ern4p is required for the UPR (13Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). Walter and co-workers (17Sidrauski C. Cox J.S. Walter P. Cell. 1996; 87: 405-413Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) demonstrated that the splicing of HAC1 pre-mRNA is not mediated by pre-mRNA processing as The results in this further this by that the sequence present in HAC1 intron is not required for the splicing (Fig. and that cleavage of the two splice sites to occur in a random (Fig. of tRNA ligase in the splicing of HAC1pre-mRNA (17Sidrauski C. Cox J.S. Walter P. Cell. 1996; 87: 405-413Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) has the that cleavage ofHAC1 pre-mRNA is also by tRNA This can be as a of in genes that encode of tRNA F. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). results revealed some important in the of of the splice sites between the tRNA and putative endonucleases responsible for the UPR. In to tRNA splicing, sequence at the splice sites of HAC1pre-mRNA to be important for cleavage (Fig. 2). In we previously showed that of two nucleotides into a between and of the 5′ splice site completely blocked the splicing (14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar), a in a of the cleavage site in the of tRNA splicing J. Cell. 1988; Full Text PDF PubMed Scopus Google Scholar). Accumulation of unfolded proteins in the ER is to be by a protein localized in the ER, and the is the and of (5Cox J.S. Shamu C.E. Walter P. Cell. 1993; 73: 1197-1206Abstract Full Text PDF PubMed Scopus (950) Google Scholar, 6Mori K. Ma W. Gething M.J. Sambrook J. Cell. 1993; 74: 743-756Abstract Full Text PDF PubMed Scopus (655) Google Scholar, C.E. Walter P. J. 1996; PubMed Scopus Google Scholar). this was in and Walter C. Walter P. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) that the C-terminal portion of the and the in sequence to mammalian has activity that HAC1pre-mRNA in The on the splicing of a point mutation introduced at of the 5′ or 3′ splice site are with the results shown in Fig. In results of the swapping (Fig. the of the in the cleavage of the two splice analysis in the cleavage sites the 5′ by nucleotide from we by mutational analysis in (14Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar) for both 5′ and 3′ splice that the cleavage between and (see Fig. 2). The for this to be In results a mechanism for sequence-specific and cleavage of the splice We and for