From the HSP Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan
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ABSTRACT |
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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 cerevisiae
and 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 of
HAC1 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.
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INTRODUCTION |
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Accumulation of unfolded proteins in the endoplasmic reticulum (ER)1 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 (1-4). 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 (5-8) as well as in mammalian cells (9-12), 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
(hac1) are unable to induce transcription of any of the target genes of the UPR and exhibit sensitivity to ER stress (7, 8,
13). 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 (13, 14). 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. 1A). 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 (13) 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 (14).
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. 15 and
16). Walter and co-workers (17) further showed that splicing of
HAC1 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
(prp2ts and prp8ts), 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 of
HAC1 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 of
HAC1 mRNA will be discussed in relation to the features
known for conventional pre-mRNA splicing as well as tRNA
splicing.
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EXPERIMENTAL PROCEDURES |
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Strains and Microbiological Techniques--
The yeast strain
used in this study was KMY1145 (MAT leu2-3, 112 ura3-52
his3-
200 trp1-
901 lys2-801 hac1
::TRP1
ura3-52::URA3-UPRE-CYC1-lacZ) (14). The
composition of synthetic complete medium used for selection of
transformants such as SC(-Ura, Leu) has been described (18).
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 (19) .
Construction of Plasmids--
Recombinant DNA techniques were
carried out as described (20). The parental single-copy plasmids
carrying the HAC1 gene, YCp-HAC1WT, and
YCp-HAC1WT(XbaI), were described previously (14). Plasmids
with some mutated HAC1, YCp-HAC1-mBp and YCp-HAC1-SL3 (Fig. 1), were constructed by site-directed mutagenesis (21). Other
mutations were introduced into YCp-HAC1WT(XbaI) by replacing the 0.18-kb XbaI640-HindIII818 or 0.11-kb
HindIII818-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-- Northern blot hybridization analysis was carried out as described previously (6, 7, 14). The positions of probes specific for the first or second exon of HAC1 are illustrated in Fig. 1A.
-Galactosidase Assays--
Cellular UPR activity was
monitored by measuring the level of
-galactosidase expressed from
the UPRE-CYC1-lacZ reporter gene that had been integrated
into the chromosome of KMY1145. Induction of
-galactosidase by
tunicamycin entirely depends on both the splicing of HAC1
pre-mRNA induced by the signaling from the ER and the direct
interaction between Hac1p thus produced and cis-acting unfolded protein-response element (UPRE) (7, 14). Assays for
-galactosidase activity in yeast were carried out as described previously (6).
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RESULTS |
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The Branchpoint-like Sequence Present in HAC1 Intron Is Not
Required for the Splicing--
We utilized the mfold server by Zuker
and Turner on the internet2
to obtain a possible secondary structure of HAC1
pre-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. 1B, 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 HAC1
intron (17) 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 intermediate during the first step in conventional
pre-mRNA splicing and is also important for formation of splicing
complex as well as commitment complex (Ref. 22 and references therein).
The Bp-like sequence in HAC1 intron is found 28 nucleotides
upstream of the 3
splice sites, which matches well with Bp sequences
usually located 20-40 nucleotides upstream of the 3
splice sites
(15).
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Sequence-specific Cleavage of the Splice Sites--
Next we
examined whether nucleotide sequences around the splice sites were
specifically recognized by putative endonucleases during the splicing
reaction. To introduce various point mutations at the 5 or 3
splice
site, we carried out PCR-mediated mutagenesis using a mutant version of
HAC1 designated YCp-HAC1WT(XbaI) as described
under "Experimental Procedures." Although
YCp-HAC1WT(XbaI) contained an XbaI site at
nucleotide 640 that changed Leu214-Asp215 to
Ser-Arg, the two amino acid changes did not affect the level of either
HAC1 mRNA or Hac1p but only slightly reduced the
transcriptional activator activity of Hac1p (14). A point mutation was
introduced into each of the seven nucleotides predicted to form a loop
structure of SL1 (5
splice site) or SL4 (3
splice site) and one
flanking nucleotide each at both sides, where G was changed to C, A was changed to T, and vice versa so that the GC content was not altered (Fig. 2). For convenience, nucleotides at the 5
side or 3
side of the
cleavage site were minus- or plus-numbered. After introduction of each
point mutant into the hac1
strain and after 3 h of
incubation in the presence or absence of tunicamycin,
-galactosidase
activity expressed from the reporter gene was determined, and the
extent of induction was compared with that of the wild-type (Fig. 2, A and B). Some of the point mutations were found
to strongly inhibit the induction, whereas others were not.
Interestingly, four out of the nine nucleotides (
3,
1, +3, and +5,
boxed in Fig. 2) conserved between the 5
and 3
splice sites were most
critical for the UPR; a point mutation of any of these nucleotides
abolished induction of
-galactosidase almost completely, with one
exception that affected the +5 nucleotide of the 3
splice site. On the other hand, the nucleotide at +2 of either splice site seemed not to be
important, and the effects of point mutations at
4,
2, +1, and +4
varied considerably between the 5
and 3
splice sites. These results
indicated that sequence integrity of the splice sites is highly
important for the UPR and suggested that putative endonucleases
specifically recognize nucleotide sequences at both of the splice
sites.
Cleavage of the 5 and 3
Splice Sites Can Occur
Independently--
Northern blot hybridization analysis was carried
out to determine which step of the splicing was blocked by point
mutations introduced at the 5
or 3
splice site (Fig.
3). Total RNAs isolated from
transformants cultured for 1 h in the presence or absence of
tunicamycin were analyzed using a probe specific for either the first
exon or the second exon of HAC1. In hac1
cells
carrying YCp-HAC1WT(XbaI), constitutively expressed 1.4-kb
pre-mRNA (lanes 1 and 13) was spliced by
tunicamycin treatment to produce 1.2-kb mature mRNA (lanes
2 and 14). In this experiment, a significantly increased amount of 0.7-kb band was detected with probe specific for
the first exon (lane 2) as compared with the previous
experiment using YCp-HAC1WT (a band marked by an asterisk in
Fig. 1C). Because this band was not detected using probe
specific for the second exon (Fig. 3, lane 14) or intron
(data not shown), it must consist only of the first exon. Similarly, a
band of 0.4 kb detected with probe specific for the second exon
(lane 14) must consist only of the second exon, whose amount
was significantly higher than that obtained with YCp-HAC1WT (data not
shown). Therefore, the increased amounts of these intermediates are
likely to result from slightly inefficient ligation of the two exons
due to the nucleotide alteration introduced to create an
XbaI site at nucleotide 640, 16 nucleotides upstream of the
5
splice site. Alternatively, the alteration introduced might have
affected RNA stability.
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Swapping the Nucleotides at the 5 and 3
Splice Sites Inhibits
Ligation but Not Cleavage--
Finally, we made three additional
mutant versions of HAC1 by swapping the nucleotides around
the 5
splice site and those around the 3
splice site, and examined
their effects on the UPR (Fig. 4). As
already shown in Fig. 2, not only seven nucleotides predicted to be
localized in the respective loop structure, but also two flanking
nucleotides at the position of
4 and +5 seemed to be important for
the cleavage. Especially, the G to C mutation at +5 of the 5
splice
site completely blocked the cleavage at the 5
splice site, whereas the
same mutation at +5 of the 3
splice site affected the splicing only
weakly. Therefore, we swapped a total of nine nucleotides to see
whether these differential effects are indicative of the involvement of
two different endonucleases in cleaving two splice sites or not.
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DISCUSSION |
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In addition to autocatalytic self-splicing of group I and group II
introns, two types of protein-mediated RNA splicing, namely spliceosome-dependent pre-mRNA splicing and tRNA
splicing, have gained much attention and which have revealed completely
different mechanisms. In conventional pre-mRNA splicing, sequences
at the 5 and 3
splice sites are strictly conserved (GT-AG or AT-AC) and sequence-specific cleavage at the splice sites is achieved by
involvement of various small nuclear RNAs and numerous proteins complexed in a spliceosome. In addition, cleavage occurs in two sequential steps. First, cleavage of the 5
splice site occurs in
concert with the formation of 2
-5
phosphodiester bond between the
5
-end of the intron and the adenosine in the Bp sequence located
upstream of the 3
splice site, thus producing a lariat structure.
Second, cleavage of the 3
splice site occurs, leading to release of
the lariat intron and simultaneous ligation of the two exons (15, 16, 24 and references therein). On the other hand, tRNA splicing is
catalyzed by the sequential action of three protein enzymes: a
site-specific endonuclease, a tRNA ligase, and a phosphotransferase. In
contrast to conventional pre-mRNA splicing, nucleotide sequences at
the splice sites in tRNAs are not conserved, and the precise cleavage
at the two splice sites is explained by a "ruler mechanism" in
which a fixed distance to the splice sites is measured from a certain
position in the mature domains of the tRNAs. Furthermore, the two
splice sites are cleaved independently (25, 26 and references therein).
Recent work conducted in two laboratories established that the regulated splicing of mRNA encoding transcription factor Hac1p/Ern4p is required for the UPR (13, 14). Walter and co-workers (17) demonstrated that the splicing of HAC1 pre-mRNA is not mediated by spliceosome-dependent pre-mRNA processing as mentioned earlier. The results reported in this paper further substantiate this notion by demonstrating that the Bp-like sequence present in HAC1 intron is not required for the splicing (Fig. 1) and that cleavage of the two splice sites appeared to occur in a random order (Fig. 3).
Direct involvement of tRNA ligase in the splicing of HAC1
pre-mRNA (17) has evoked the possibility that cleavage of
HAC1 pre-mRNA is also catalyzed by tRNA endonuclease.
This hypothesis can now be pursued as a result of recent success in
cloning genes that encode subunits of tRNA endonuclease (26). However,
our results revealed some important differences in the mode of
recognition of the splice sites between the tRNA endonuclease and
putative endonucleases responsible for the UPR. In contrast to tRNA
splicing, sequence integrity at the splice sites of HAC1
pre-mRNA appeared to be very important for cleavage (Fig. 2). In
addition, we previously showed that insertion of two nucleotides into a
position between 2 and
1 of the 5
splice site completely blocked
the splicing (14), whereas a similar insertion should result in a
predictable shift of the cleavage site in the case of tRNA splicing
(27).
Accumulation of unfolded proteins in the ER is considered to be sensed
by Ire1p/Ern1p, a transmembrane protein kinase localized in the ER, and
the signal is transmitted across the lipid bilayer through
oligomerization and autophosphorylation of Ire1p (5, 6, 28). While this
paper was in review, Sidrauski and Walter (29) reported that the
purified C-terminal portion of Ire1p containing the kinase domain and
the tail domain similar in sequence to mammalian RNase L has
endonuclease activity that successfully cleaves HAC1
pre-mRNA in vitro. The effects on the splicing of a
point mutation they introduced at 1 of the 5
or 3
splice site are
consistent with the results shown in Fig. 3. In addition, our results
of the swapping experiment (Fig. 4) may support the involvement of the
Ire1p homodimer in the cleavage of the two splice sites. However, their
primer extension analysis in vitro shifted the actual
cleavage sites toward the 5
side by one nucleotide from those we
determined by mutational analysis in vivo (14) for both 5
and 3
splice sites; namely, they claim that the cleavage occurs
between
2 and
1 (see Fig. 2). The reason for this apparent discrepancy remains to be clarified.
In conclusion, our results reported here unraveled a novel mechanism for protein-mediated RNA splicing: sequence-specific and non-sequential cleavage of the splice sites.
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ACKNOWLEDGEMENTS |
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We thank Masako Nakayama, Mayumi Ueda, and Hideaki Kanazawa for technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-75-315-8656;
Fax: 81-75-315-8659; E-mail: kazumori{at}hsp.co.jp.
1 The 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.
2 http://www.ibc.wustl.edu/~zuker/rna.
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REFERENCES |
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