From the Department of Biological Chemistry and
Molecular Pharmacology and ¶ Center for Blood Research and
Department of Pathology, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, November 27, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Eukaryotic mRNA capping enzymes are
bifunctional, carrying both RNA triphosphatase (RTPase) and
guanylyltransferase (GTase) activities. The Caenorhabditis
elegans CEL-1 capping enzyme consists of an N-terminal region
with RTPase activity and a C-terminal region that resembles known
GTases, However, CEL-1 has not previously been shown to have GTase
activity. Cloning of the cel-1 cDNA shows that the
full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not
originally predicted from the genomic sequence. Full-length CEL-1 has
RTPase and GTase activities, and the cDNA can functionally replace
the capping enzyme genes in Saccharomyces cerevisiae. The
CEL-1 RTPase domain is related by sequence to protein-tyrosine
phosphatases; therefore, mutagenesis of residues predicted to be
important for RTPase activity was carried out. CEL-1 uses a mechanism
similar to protein-tyrosine phosphatases, except that there was not an
absolute requirement for a conserved acidic residue that acts as a
proton donor. CEL-1 shows a strong preference for RNA substrates of at
least three nucleotides in length. RNA-mediated interference in
C. elegans embryos shows that lack of CEL-1 causes
development to arrest with a phenotype similar to that seen when RNA
polymerase II elongation activity is disrupted. Therefore, capping is
essential for gene expression in metazoans.
Most eukaryotic and viral mRNAs are modified at their 5' end
by a "cap" structure that consists of a 7-methylguanosine moiety attached to the 5' terminus via a 5'-5' linkage (1). Three sequential
enzymatic activities are required to form the "cap 0" structure,
m7GpppN. First, an RNA 5'-triphosphatase
(RTPase)1 removes the
Previously we characterized a putative capping enzyme gene, which we
named cel-1, that emerged from the Caenorhabditis
elegans genome sequencing project (3). The open reading frame
(ORF) of this gene originally predicted by the Nematode Sequencing
Project was 573 amino acids. The C-terminal 340 amino acids exhibit
very strong similarity to yeast and viral GTases. The N-terminal region has significant sequence similarity to the protein-tyrosine phosphatase (PTP) family, including the active site consensus motif
(I/V)HCXXGXXR(S/T)G (4-7). We proved that the
isolated N-terminal region (residues 1-236) of CEL-1 has RTPase
activity (3, 8). However, we were unable to demonstrate that the
C-terminal region had GTase activity.
The ORF used in the earlier study was based on predictions of exons
within genomic sequence. Since then the C. elegans Expressed Sequence Tag (EST) data base has produced several cDNA sequences predicted to encode a longer form of CEL-1 that has additional residues
at both the N and C termini. Protein produced from the longer ORF fully
substitutes for the Saccharomyces cerevisiae GTase and
RTPase in vivo. The longer CEL-1 C-terminal domain has GTase
activity in vitro. We further characterized the isolated RTPase domain both in vivo and in vitro. We
analyzed its catalytic properties, including the effect of RNA chain
length on the activity. Mutagenesis confirms a mechanistically
conserved role for key residues found in both the RTPase and PTPs.
Surprisingly, the CEL-1 RTPase did not require linkage to the GTase for
targeting to pre-mRNA in S. cerevisiae. Finally, we used
RNA-mediated interference (RNAi) to demonstrate that CEL-1 is essential
in vivo for development of C. elegans.
DNA Cloning Methods--
Supplementary tables listing
oligonucleotides and plasmids used in this study are available at the
Buratowski laboratory web site.2 PCR for construction
of plasmids and site-directed mutagenesis were carried out with Vent
DNA polymerase (New England BioLabs).
Cloning of CEL-1 cDNA--
To obtain a full-length cDNA
for CEL-1, a two-step Mega-Primer PCR was performed with two EST
clones, yk786d02 and yk798b08 (supplied by Dr. Y. Kohara, National
Institute of Genetics, Mishima, Japan). In the first reaction, a 1.2-kb
fragment was amplified from the 5' region of CEL-1 using
CEL-1upstreamA and Cel1-4 as primers and yk786d02 as template. The
product was purified and used in the second PCR as a megaprimer. The
second reaction used yk798b08 as template and used 3'CEL-1longer and
CEL-1upstreamA as primers for secondary amplification. A 1.8-kb product
was subcloned into pCR-Script SK(+) (Stratagene) to produce pBS-CEL-1.
To create CEL-1-(1-585), 3'CEL-1longer
was replaced with Celeg. CE-C.
Genetic Manipulation of S. cerevisiae--
S.
cerevisiae strains used in this study were YSB244 (MATa
ura3-52 leu2-3,112 his3 Yeast Whole-cell Extract Preparation and Protein
Analysis--
Whole-cell extracts from S. cerevisiae,
immunoprecipitation, and subsequent enzyme-GMP formation assay were as
previously described (11).
Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out using either single-stranded phagemid (15, 16) or PCR (17).
In both strategies, the plasmid pBS-CEL-1-(13-585) (Ref. 3, listed as
pBS-CEL-1 therein) was used as the template. Mutations were verified by dideoxy-DNA sequencing.
To change Arg-142 to Lys (R142K) or Ala (R142A), Asp-76 to Asn (D76N),
and Glu-111 to Gln (E111Q), phagemid mutagenesis was performed with
mutagenic oligonucleotides CEL1-R142K, CEL1-R142A, CEL1-D76N, or
CEL1-E111Q. The resulting plasmids, pBS-CEL-1 R142K, pBS-CEL-1 R142A,
pBS-CEL-1 D76N, and pBS-CEL-1 E111Q, served as template for subsequent
PCR. Except for D76N, we amplified 0.7-kb fragments corresponding to
RTPase domain containing those mutations using Celeg. CE-B and
CEL-1T222stop as primers. For the wild type and the active site
cysteine mutants, we used pBS-CEL-1-(13-585), pET-his7CEL-1-(13-248)C136S, and pET- his7CEL-1-(13-248)C136A as
templates for the same PCR. Each product was subcloned into pCR-Script
SK(+) to generate pBS-CEL-1-(13-221) version 2, pBS-CEL-1-(13-221)C136S version 2, pBS-CEL-1-(13-221)C136A version 2, pBS-CEL-1-(13-221)R142K version 2, pBS-CEL-1-(13-221)R142A version 2, and pBS-CEL-1-(13-221)E111Q version 2, respectively. We further
subcloned inserts from these plasmids into yeast expression vector pAD5
(11) and bacterial expression vector pSBET-His7 (18). For
D76N, a 0.7-kb fragment was amplified with pBS-CEL-D76N as template and
Celeg. CE-B and CEL1(3'Sac) as primers. The product was digested with
NcoI and SacI and subcloned into pAD5.
PCR-mediated site-directed mutagenesis was used to change Asp-112 to
Asn (D112N). In the first reaction, a 0.3-kb fragment was amplified
with CEL-1D112N and CEL-1T222stop. In the second reaction, primers were
Celeg. CE-B and the product from the first reaction as a megaprimer.
The product was subcloned onto pCR-Script SK(+) to produce
pBS-CEL-1-(13-221)D112N.
Recombinant Protein Production and Purification--
Mouse
capping enzyme (MCE) full-length protein and the RTPase domains of MCE
and CEL-1 were expressed using a T7 promoter/polymerase system (19).
Escherichia coli strain BL21(DE3) was transformed with the
appropriate expression plasmids and cultured in 500-ml media at
37 °C to an A600 = 0.5. The proteins
were induced as described (18). All further operations were at
0-4 °C. Lysate was prepared by sonication in buffer B (50 mM Tris-HCl, pH 7.9, 10% (v/v) glycerol, 1 mM
phenylmethylsulfonyl fluoride) with 300 mM KCl and 0.5%
Nonidet P-40. After incubating soluble extracts (100,000 × g supernatant fraction) with 2 ml of nickel nitrilotriacetic acid-agarose resin (Qiagen) for 2 h on a rotator, the resin was poured into a column (1.5 × 2.5 cm) and extensively washed with buffer B with 20 mM KCl and 25 mM imidazole.
Bound proteins were eluted with buffer B containing 20 mM
KCl and 600 mM imidazole and immediately supplemented with
1 mM EDTA and 1 mM DTT. Proteins were further
purified by chromatography with a column (1.2 × 9.0 cm) of
heparin-Sepharose CL-6B (Amersham Biosciences). After washing the resin with 20 mM KCl in buffer C (20 mM
Tris-HCl, pH 7.9, 1 mM EDTA, 1 mM DTT, 10%
(v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride),
proteins were eluted with a 100-ml linear gradient of 20-300
mM KCl in buffer B. Purified proteins were visualized by
SDS-PAGE and Coomassie Brilliant Blue staining and by immunoblot analysis with monoclonal anti-polyhistidine antibody
(anti-His6, Clontech).
Preparation of Substrate for RTPase
Assay--
[ Enzymatic Assays--
GTase, RTPase, and nucleotide
phosphohydrolase activities were assayed as previously described (3, 8,
18).
RNAi Analysis--
In vitro synthesized
double-stranded (ds)RNA (Megascript, Ambion) was produced using
pBS-CEL-1-(13-221) and pBS-CEL1-(1-558) and injected at 1.0 µg/µl
into young adults (2-8 fertilized embryos). Equivalent results were
obtained with cel-1 RNAs covering the RTPase or GTase
domains. ama-1(RNAi) and cel-1(RNAi) embryos were analyzed at 24 and 36 h post-injection, respectively, when uniform populations of arrested embryos appeared. For immunostaining, embryos
were collected from dissected hermaphrodites 36 h after injection.
Because most analyses were performed before terminal embryonic
developmental arrest, RNAi effectiveness was confirmed by monitoring
sibling embryos that were allowed to develop.
Immunostaining--
For Detection of GTase Activity in C. elegans Nuclear Extract--
To
determine the gel mobility of the native capping enzyme, we incubated
C. elegans nuclear extract with [ Identification of an Extended C. elegans Capping Enzyme Open
Reading Frame--
Previously, we (3) and others (28) found that the
predicted gene C03D6 (GenBankTM accession number Z75525)
from the C. elegans genome sequencing project had
significant similarity to the yeast GTase. Similar capping enzyme
genes from other metazoans were described (29-33). All of these
proteins contain motifs found in the GTase proteins/domains of yeast
and viruses (2, 28, 34-36; Fig.
2A) as well as an N-terminal domain related to the PTPs.
The protein encoded by the ORF for CEL-1 is somewhat shorter at the C
terminus than its homologues from other species (Fig. 2A).
Using the predictions of exon structure, we amplified a CEL-1 ORF from
C. elegans cDNA. Although we could demonstrate RTPase activity of the N-terminal domain, we were not able to show GTase activity of the C-terminal domain. Therefore, we searched the EST data
base (www.ncbi.nlm.nih.gov/BLAST/) for naturally occurring CEL-1
cDNAs and found SP9F10 (accession number BE228078). This cDNA
encodes an ORF containing the previously predicted amino acids 534-573
of CEL-1 but which has an extra 38 residues at the C terminus due to a
splicing event that was not predicted from the genomic sequence. The
C. elegans EST data base server at the DNA Data Bank of
Japan (www.ddbj.nig.ac.jp/c-elegans/html/CE_BLAST.html) contains clone yk786d02 containing sequences for the 5'
region of CEL-1. This encodes the previously identified RTPase domain (Refs. 3 and 8; designated residues 1-236 in those papers). However,
there is an in-frame initiation codon 36-base upstream of the one
previously believed to be the translation start site (Ref. 3; Fig.
2B). Combining this new cDNA information, full-length CEL-1 is predicted to have an additional 12 residues at the N termini
and 38 residues at the C termini, for a total of 623 amino acids. The
predicted molecular mass is 72 kDa, in good agreement with the size of
the GTase detected in worm extract (Fig. 1). We therefore have
renumbered the CEL-1 amino acids, and the previously analyzed shorter
protein (3, 8) will herein be referred to as CEL-1-(13-585).
CEL-1 Is a Bifunctional Capping Enzyme with Both RTPase and GTase
Activities--
Unlike the metazoan enzyme, capping enzyme in the
yeast S. cerevisiae is a complex of RTPase and GTase
subunits (37). These polypeptides are encoded by the CET1
and CEG1 genes, respectively, both of which are essential
for cell viability (38, 39). Ceg1 is related by sequence to the viral
and metazoan GTases (2, 36). In contrast, Cet1 is not related to PTPs
or metazoan RTPase domains (40).
We tested whether CEL-1 can function in place of CEG1 and
CET1 in S. cerevisiae (Fig.
3A). The
ceg1
A truncation mutant of MCE containing only the GTase domain (residues
211-597) can replace CEG1 in S. cerevisiae (41).
The corresponding domain of CEL-1 (residues 221-623, see Fig.
2A) supports viability (Fig. 3B) of the
ceg1 The CEL-1 RTPase Domain Can Function Independently of the GTase
Domain in Vivo--
We previously showed that CEL-1-(13-248) has
RTPase activity in vitro (3, 8). Although full-length CEL-1
can simultaneously replace yeast Ceg1 and Cet1, we tested whether
overexpression of CEL-1 derivatives could rescue cet1
To avoid the complications of Ceg1 dependence upon Cet1 interaction,
the CEL-1 derivatives were assayed in cells expressing GTases from
other organisms. In ceg1
To determine whether the mouse and C. elegans RTPase domains
were interacting with the GTases or, instead, were independently acting
at mRNA 5' ends, immunoprecipitation experiments were carried out.
Epitope-tagged Cet1, CEL-1-(13-221), or MCE-(1-210) were co-expressed
with either pce1 or Cgt1. The RTPases were immunoprecipitated with
12CA5 (Fig. 4C). The top panel shows that each of
the RTPases was expressed and immunoprecipitated efficiently. The
precipitate was assayed for guanylylation with
[
Presumably, CEL-1-(13-221) and MCE-(1-210) do not support
growth of a cet1 Mutation Analysis of CEL-1 RTPase Domain--
Members of the PTP
superfamily, including the metazoan capping enzyme RTPases, have an
active site consensus sequence of
(I/V)HCXXGXXR(S/T)G. The nucleophilic cysteine
attacks the phosphate. The arginine residue contributes to
transition-state stabilization via the formation of hydrogen bonds with
two oxygens of the phosphate (47). Outside the active site motif, a
conserved aspartic acid residue serves to stabilize the leaving group
(48-50). In the PTPs, this acidic residue is believed to act as a
general acid, donating a proton to the leaving group oxygen of the
substrate tyrosine residue.
To examine the degree of mechanistic conservation between the PTPs and
RTPases, we analyzed CEL-1 derivatives mutated at conserved residues
important for the PTP mechanism. Arginine 142 in the consensus motif
was mutated. Also, aspartate 76, glutamate 111, and aspartate 112 were
mutated because they were candidates for the proton-donating acidic
residue. Arg-142 and Asp-76 are conserved in all of the PTP-like
RTPases, whereas Glu-111 and Asp-112 are not (Fig. 2B). The
histidine-tagged mutants C136S, R142K, D76N, E111Q, and D112N were
purified from E. coli (Fig.
6A). Their RTPase activities
were tested with [
We also tested if these mutants can support viability of a
ceg1
Residues Glu-111 and Asp-112 are not highly conserved and do not seem
to be vital in vivo (Fig. 6C). In contrast,
Asp-76 is conserved, and a D76N mutant cannot support viability in
yeast. However, the D76N mutant still has partial activity in
vitro, indicating that the carboxylate side chain is not
absolutely required. We speculate that the reduced activity of D76N is
not sufficient to rescue cells in the heterologous yeast system.
Mutation of the equivalent residue in MCE or BVP (Asp-66 of MCE and
Asp-60 of BVP) only slightly diminished the activity (51-53).
Therefore, in contrast to the PTPs, general acid catalysis may not be
essential for the mechanism of the RNA phosphatases.
Effect of Chain Length of RNA on RTPase Domain of Metazoan Capping
Enzyme--
The DNA primase of bacteriophage T7 uses a DNA template to
make short RNA primers (2-10 nucleotides) that begin with the sequence pppApC (21). Using this system, substrates of various sizes were
prepared for RTPase assays. We previously reported that CEL-1-(13-248) efficiently uses a trinucleotide substrate (pppApCpC) but not a
mononucleotide (pppA) (8). Here, we prepared dimer (pppApC), trimer
(pppApCpC), tetramer (pppApCpCpC), and pentamer (pppApCpCpCpC) RNAs (Fig. 7A). First, the
activity of CEL-1-(13-221) was tested with dinucleotide labeled at
either the
The pH optimum of the RTPase reaction was about pH 8.0, and the
reaction was severely inhibited below pH 7.0 (data not shown). Sodium
vanadate is an inhibitor of PTPs that acts as a transition-state mimic
(54). Vanadate also inhibited CEL-1-(13-221), with 60% inhibition
observed at 1 µM (data not shown). Like PTPs, CEL-1 RTPase is independent of, and in fact inhibited by, divalent cations (data not shown). Similar inhibition was reported for MCE, PIR1, and
BVP (51, 55).
Next we tested the effect of RNA chain length (Fig.
8). CEL-1-(13-221) hydrolyzes the
CEL-1 Is Essential for Embryonic Development and CTD Ser-2
Phosphorylation--
To investigate the requirement for CEL-1 in
vivo, we used RNAi to inhibit its expression during embryogenesis
(56). The early embryo provides an advantageous system for analyzing
functions of essential transcription or mRNA processing machinery
components in vivo. The initial stages of C. elegans development are orchestrated by maternally derived
proteins and mRNAs, making it possible for embryos to survive until
approximately the 100-cell stage when new mRNA transcription is
prevented (27, 57).
cel-1(RNAi) embryos arrested development after
forming ~100 cells that lacked any signs of differentiation (Fig.
9A). This terminal arrest
phenotype is very similar to that observed when the pol II large
subunit or various other broadly essential mRNA transcription
factors are inhibited by RNAi (23, 24, 27, 57). However, early cell
division timing and cleavage planes were normal in
cel-1(RNAi) embryos, suggesting that these
embryos contained appropriate maternal mRNA stores (not shown). One
abnormality was the cell cycle period of the endodermal precursor cells
Ea and Ep, which was shortened compared with wild type. This particular cell cycle abnormality characteristically occurs in response to broad
defects in early embryonic transcription, including mutation or RNAi
knockdown of the C. elegans orthologs of the transcription elongation factor genes spt5 and spt6 (23, 24,
27, 57). Together, the data suggest that lack of cel-1
activity may significantly impair new embryonic mRNA
production.
To further characterize how the process of mRNA production was
affected in cel-1(RNAi) embryos, phosphorylation
of RNA polymerase II was analyzed. The CTD of the pol II large subunit
consists of repeats based in the consensus sequence YSPTSPS (42 copies in C. elegans). The CTD interacts with mRNA processing
factors, linking them to the transcribing polymerase (58, 59). Near promoters, the CTD repeat is primarily phosphorylated on serine 5 by
the transcription factor IIH kinase, recruiting mRNA capping enzyme (26, 60, 61). As pol II moves away from the promoter, the CTD
phosphorylation shifts primarily to serine 2 (60). During metazoan
transcription, CTD serine 2 is phosphorylated primarily by the kinase
P-TEFb (CDK-9/cyclin T) (23, 62). CTD Ser-5 and Ser-2 phosphorylation
can be specifically detected in embryonic nuclei by staining with the
P-CTD and H5 antibodies, respectively (26, 27, 63), which we refer to
as
In the early C. elegans embryo, the appearance of both
In cel-1(RNAi) embryos, total levels of the pol
II large subunit AMA-1 are unaffected (Fig. 9B), but CTD
phosphorylation was highly abnormal. As in wild type embryos, in
cel-1(RNAi) embryos Ser-5 phosphorylation was
detectable as bright punctate staining pattern in somatic nuclei (Fig.
9C). In contrast, in cel-1(RNAi) embryos levels of specific Here we characterize the full-length C. elegans capping
enzyme, CEL-1. Based on cDNAs in the EST databases, CEL-1 is a
623-amino acid protein with both RTPase and GTase activities. This
matches the size of the enzyme-GMP intermediate detected in C. elegans nuclear extract (Fig. 1). Interestingly, multiple
cDNAs for human and Xenopus capping enzymes have been
described, possibly produced by alternative splicing of mRNA
(31-33). These cDNA variants encode an intact N-terminal RTPase
domain but have either internal deletions or truncations in the
C-terminal GTase domain. As a result, the proteins from these variants
would only have RTPase activity. PCR analyses showed that these short
forms are expressed, but their physiological function is unknown. To
date, no cDNAs corresponding to a shortened capping enzyme have
been found in the C. elegans EST data base. CEL-1 was
originally predicted to have 573 amino acids. CEL-1-(1-585) has RTPase
activity but does not complement a S. cerevisiae GTase
mutant ceg1 The CEL-1 RTPase domain (3) was the founding member of a subfamily of
PTP-like RNA phosphatases. This subfamily includes the capping enzyme
RTPases and RNA tri- and diphosphatases, whose functions are unknown
(Fig. 2B). All members contain a nucleophilic cysteine
necessary for activity (3, 8, 41, 51, 55, 64, 65). A phosphocysteine
intermediate was detected with MCE and BVP (52, 53). Other PTP-like
enzymes with substrates other than phosphotyrosine have been reported;
these include the phosphoinositide phosphatase PTEN/MMAC1 and
myotubularin (66) and S. cerevisiae arsenite reductase Acr2
(67).
In PTPs, the formation and hydrolysis of the phosphocysteine
intermediate of PTP requires transition-state stabilization by the
arginine residue within the consensus motif (4-7). Mutagenesis of MCE
(51, 52), BVP (53), and CEL-1 (Fig. 6) shows that this residue is
essential for RNA phosphatase activity, providing further evidence that
the PTP and RNA phosphatases use the same enzymatic mechanism. On the
other hand, mutation of a conserved aspartic acid residue in the
RTPases (Asp-76 of CEL-1, Asp-66 of MCE, and Asp-60 of BVP; see Fig.
2B) only slightly diminishes activity (Fig. 6; Refs.
51-52). The equivalent mutations in PTPs lower activity by
102-105-fold (4-7). X-ray crystallography of
MCE-(1-210) shows that Asp-66 is positioned differently from the
essential general acid aspartate loop described for PTPs (52).
Apparently, the RTPase mechanism does not conserve the function of
this residue.
Both CEL-1-(13-221) and MCE-(1-210) can remove the RTPases and GTases are typically linked with each other, either on the
same protein (metazoans) or in a complex (yeast). In S. cerevisiae, the interaction between the GTase (Ceg1) and RTPase (Cet1) subunits is essential for cell viability. Cet1 cannot be replaced by the RTPase domains from MCE or CEL-1, presumably because these RTPases cannot interact with Ceg1. It was originally proposed that the primary role of the linkage between GTase and RTPase on a
single polypeptide was to guide RTPase to pol II transcription complex
(43, 64). However, both CEL-1-(13-221) and MCE-(1-210) can support
viability when Ceg1 is replaced with MCE-(211-597), S. pombe pce1, or C. albicans Cgt1 (Fig. 4, A
and B). Because we did not detect any tight
interaction between these RTPases and GTases (Fig.
4C), we conclude that the metazoan RTPase domain can be
targeted to pre-mRNA and function without any linkage to GTase. The
primary function of the Cet1 interaction with Ceg1 is instead required
for the activity of Ceg1 (10, 11). Other fungal and metazoan GTases do
not require an interaction with RTPase for activity (45, 46).
Although we found that the link between RTPase and GTase domains is not
absolutely required for the capping enzymes of metazoans or fungi other
than S. cerevisiae, this does not mean that the interaction
is unimportant. To substitute for Cet1 in vivo, it was
necessary to overexpress the isolated metazoan RTPase domain with a
strong promoter and a high copy plasmid (Ref. 76 and this study). In
contrast, a low copy plasmid of the full-length enzyme was sufficient
for rescuing a cet1 Finally, we examined the requirement for CEL-1 in vivo using
RNA-mediated inactivation of the gene.
cel-1(RNAi) embryos arrest development with a
phenotype that is characteristic of a broad transcription defect. A
similar phenotype is seen upon RNAi knockdown of ama-1 (pol
II), ttb-1 (TFIIB), or multiple TAFs (23, 24, 27). One
cel-1(RNAi) phenotype is strikingly different
from effects seen upon depletion of basal initiation factors. In those cases, levels of CTD phosphorylation at both serine 5 and serine 2 were
lowered in parallel, often reduced to undetectable levels. For example,
in ttb-1(RNAi) embryos, in which basal factor
TFIIB is knocked down, both serine 5 and serine 2 phosphorylation are reduced to background (23, 27). In cel-1(RNAi)
embryos, CTD serine 5 phosphorylation appears to be relatively
unaffected, whereas serine 2 phosphorylation is dramatically reduced
(Fig. 9B). The only other example of this "uncoupling"
of CTD serine 5 and 2 phosphorylation occurred when we depleted either
of the P-TEFb components, CDK-9, or cyclin T (23). Levels of the CDK-9 kinase appear normal in cel-1(RNAi) embryos,
however. Because serine 5 phosphorylation occurs primarily near the
promoter, the generally normal levels in
cel-1(RNAi) embryos suggest that transcription initiation may be close to normal. The markedly decreased levels of
serine 2 phosphorylation, a modification linked to elongation phase,
suggests that the absence of capping enzyme interrupts the progression
of transcription. It will be interesting to determine whether the lack
of capping enzyme decreases the efficiency with which P-TEFb or other
elongation factors are recruited to transcribed genes. This would be
the latest of many connections have recently emerged between
transcription elongation and mRNA processing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate from the 5' end of the RNA substrate to leave a
diphosphate end. Next, a GTP::mRNA guanylyltransferase (GTase) catalyzes transfer of GMP from GTP, resulting in a 5'-5' linkage, GpppNp. These two activities are typically associated and
copurify as mRNA capping enzyme. A third protein, RNA
(guanine-7-)-methyltransferase, adds a methyl group to the N-7 position
of the guanine cap (1, 2).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
200
ceg1
1::HIS3 {pRS316-CEG1})
(9), YSB533 (MATa ura3-52 leu2
1
trp1
63 his3
200 lys2
202
cet1
1::TRP1 {pRS316-CET1})
(10), and YSB719 (MAT
ura3-52 leu2
1
trp1
63 his3
200 lys2
202
cet1
1::TRP1
ceg1
3::LYS2
{pRS316-CEG1-CET1}) (11). We introduced plasmids into these strains
using a modified lithium acetate transformation protocol (12). Media
preparation, plasmid shuffling with 5-fluoroorotic acid (5-FOA), and
other yeast manipulations were carried out by standard methods (13, 14).
-32P]ATP- and
[
-32P]ATP-terminated oligoribonucleotides for the
RTPase assay were synthesized with the DNA primase protein of
bacteriophage T7 (8, 20). The standard reaction (100 µl) contained 40 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
10 mM DTT, 50 µg/ml bovine serum albumin, 50 mM potassium glutamate, 2 mM dTTP, 2 mM CTP, 0.3 mM [
-32P]ATP or
[
-32P]ATP (500-1000 cpm/pmol) (PerkinElmer Life
Sciences), 1 nM synthetic oligonucleotide template, and 1 µM (hexamer) T7 primase (21). The sequences of the
oligonucleotides used are: diribonucleotide pppApC,
5'-(C)6GTC(T)25-3'; trinucleotide
pppApCpC, 5'-(C)5GGTC(T)25-3'; tetraribonucleotide pppApCpCpC,
5'-(C)4GGGTC(T)25-3'; pentaribonucleotide pppApCpCpCpC, 5'-(C)3GGGGTC(T)25-3'. The
reaction was incubated at 37 °C overnight. After extraction
with phenol-chloroform (1:1), RNAs were precipitated with ethanol and
dissolved in 500 µl of buffer A (20 mM Tris-HCl, pH 7.9, 7 M urea) containing 100 mM NaCl. RNAs were
further purified by chromatography with a column (1.0 × 27.0 cm)
of DEAE-Sephadex A-25 (Amersham Biosciences) pre-equilibrated with buffer A with 100 mM NaCl. The column was washed with
300 ml of buffer A with 120 mM NaCl, and RNAs were eluted
with a 700-ml linear gradient of 150-350 mM NaCl in buffer
A. RNAs were analyzed with electrophoresis on a 36% polyacrylamide, 3 M urea gel and autoradiography. Samples were then pooled
and dialyzed against 10 mM Tris-HCl, pH 8.0, 1 mM EDTA with Spectra/Por 7 (Spectrum). The RNAs were then
lyophilized and dissolved in water. Previously, we used triethylamine
bicarbonate (TEA-HCO
-32P]GTP-terminated RNA was synthesized by
in vitro transcription of linearized plasmid template (3)
using recombinant polyhistidine-tagged T7 RNA polymerase (22).
-CDK-9 (23, 24) and
-pol II (25)
staining, embryos were subjected to 2% paraformaldehyde fixation and
freeze-cracked before treating with methanol. Washes and antibody
incubations were performed in PBT (1× phosphate-buffered saline, 1%
Triton X-100, 1% bovine serum albumin) before staining. Staining with the phosphorylated C-terminal domain (CTD-P) (
-Ser(P)-5) (26) and H5 (
-Ser(P)-2) (Berkeley Antibody Co.) was performed as in Walker et al. (27). Images were captured with a Zeiss
AxioSKOP2 microscope and AxioCam digital camera, and antibody staining
intensities were compared over a range of exposure times. Pixel
intensities were standardized using Adobe Photoshop 6.0.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP
to form the covalent GTase-[32P]GMP intermediate (Fig.
1). The major labeled band was about 70 kDa (lane 2). Complex formation was dependent on the
presence of divalent cation (lanes 3 and 4) and
specific to guanine nucleotide (lanes 5 and 6).
The reaction reached completion within a short period of time at
0 °C (lane 7). Therefore, the C. elegans
capping enzyme appears to be a single protein of ~70 kDa.
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Fig. 1.
Detection of the capping enzyme-GMP covalent
intermediate in C. elegans nuclear extract. 3 µM [ -32P]GTP was incubated at 30 °C
for 10 min with 20 µg of C. elegans nuclear extract
protein in 20 mM Tris-Cl, pH 7.5, 2 mM
MnCl2, 5 mM DTT (lanes 2-7).
Lane 1 is a control without protein. MnCl2 was
omitted in lanes 3 and 4, and an additional 2 mM EDTA was added in lane 4. Lane 5 is the same as lane 2 except 30 µM unlabeled
ATP was added. In lane 6, 30 µM unlabeled GTP
was added. In lane 7, the reaction mixture was incubated on
ice instead of 30 °C for 10 min. After the reaction, proteins were
separated by SDS-PAGE, and proteins covalently bound to GMP were
visualized by autoradiography.
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Fig. 2.
Protein sequence alignment of CEL-1
with capping enzymes from various species. Similarity analysis was
carried out on the National Center for Biotechnology Information Web
server using the BLAST algorithm (78). Sequence alignments were made
using SEQVU. Letters represent the single-letter amino acid
code, and numbers represent the positions of the amino acid
residues. Boxed residues denote identities, and shaded
residues signify similarity to the CEL-1 amino acid sequence.
A, alignment of C-terminal GTase regions. The following
amino acid sequences are shown: CEL-1 (this study); xCAP1 (AF218793,
residues 207-598 of Xenopus laevis capping
enzyme (33)); MCE (AF025653, residues 211-597 of mouse capping enzyme
(29, 30); HCE (AB009022, residues 211-597 of human capping enzyme
(29); Drosophila (AE003495, residues 223-649 of a
Drosophila melanogaster open reading frame);
Arabidopsis (AC009326, residues 294-653 of an
Arabidopsis thaliana open reading frame). The
asterisk shows the active site lysine residue (36). Motifs
that are highly conserved in GTases (36) are designated by bars
above and below the sequences. The arrow
shows the position of the previously predicted stop codon of CEL-1 (3).
B, alignment of N-terminal RTPase regions. The following
deduced amino acid sequences are shown: CEL-1 (this study); xCAP1
(AF218793, residues 1-206); MCE (AF025653, residues 1-210); HCE
(AB009022, residues 1-210); Drosophila (AE003495, residues
1-222); Arabidopsis (AC009326, residues 80-293). The amino
acid sequences of two PTP-like RNA phosphatases are also shown, human
PIR1 (AF023917, residues 31-169 (79)) and baculovirus BVP (L22858
(80)). The PTP active site consensus motif is marked by a
line. Arrows and asterisks show the
residues of CEL-1 mutated in this study. The diagonal arrow
shows the position of the initiation methionine previously reported
(3).
cet1
strain YSB719 (11) was transformed
with a high copy plasmid expressing HA epitope-tagged CEL-1 from the
constitutive ADH1 promoter. An expression construct for the
MCE was used as a positive control. After shuffling out the
CEG1/CET1 plasmid with 5-FOA, growth was observed
with cells expressing either MCE or CEL-1. In contrast, expression of
CEL-1-(1-585) did not rescue cells. Therefore, CEL-1 with the extended
C terminus, but not the shorter form previously analyzed, has both
capping enzyme RTPase and GTase activities.
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Fig. 3.
The CEL-1 GTase domain can replace Ceg1 in
S. cerevisiae. A, complementation of a
double deletion mutant (ceg1 cet1
) lacking
both the GTase and RTPase. The CEG1/CET1
shuffling strain YSB719 (11) was transformed with the following
LEU2/2-µm plasmids: vector (pAD5); MCE
(pGL-MCE, expressing mouse capping enzyme MCE1 from the GPD
promoter); CEL-1 (pAD5-CEL1, expressing CEL-1 from the
ADH1 promoter); CEL-1-(1-585) (pAD5-CEL-1-(1-585),
expressing residues 1-585 of CEL-1 from the ADH1 promoter).
Leu+ transformants were tested for growth after shuffling
out pRS316-CEG1-CET1 (11). B, complementation of
ceg1
by the C-terminal region of CEL-1. The
CEG1 shuffling strain YSB244 (9) was transformed with
following LEU2/2 µm plasmids: vector (pAD5);
CEG1 (pAD5-CEG1); MCE-(211-597)
(pAD5-MCE-(211-597) (11)); and CEL-1-(221-623)
(pAD5-CEL-1-(222-623), expressing CEL-1-(222-623) from the
ADH1 promoter). Leu+ transformants were tested
for growth after plasmid shuffling (9). C, formation of a
CEL-1-GMP intermediate. 10 µg of yeast whole cell extract protein was
incubated for 10 min at 30 °C with 3 µM
[
-32P]GTP. After the reaction, the proteins were
separated by SDS-PAGE and analyzed by autoradiography. Extracts were
prepared from YSB719 transformed with pAD5 (left panel,
lane 1) or pAD5-CEL-1 (left panel, lane
2) and YSB244 transformed with pAD5 (right panel,
lane 1), pAD5-CEG1 (right panel, lane
2) or pAD5-CEL-1 (222-628) (right panel, lane
3). Note that at this exposure the endogenous Ceg1 is not
visible.
strain YSB244 (9). Whole-cell extracts from yeast
expressing CEL-1 were assayed for capping enzyme-GMP complex (Fig.
3C). A protein of about 70 kDa was detected (left
panel, lane 2), the same size as that of the complex
detected in C. elegans nuclear extract (Fig. 1). We also assayed extracts from yeast expressing either epitope-tagged
CEL-1-(222-623) or CEL-1-(222-585) for the presence of protein by
immunoblot using anti-HA monoclonal antibody 12CA5 (42) and for
enzyme-GMP complex formation. Both proteins were detected in the
immunoblot (data not shown). In contrast, GTase activity was detectable
with CEL-1-(222-623) but not CEL-1-(222-585) (Fig. 3C,
right panel, lane 3 and data not shown). These
results again show that the residues 586-623 are important for CEL-1
GTase activity.
strain YSB533 (10) Neither CEL-1 derivatives 1-221, 13-221, nor
1-585 could replace CET1 (Fig.
4B and data not shown).
However, this was not surprising because GTase activity was being
supplied by Ceg1. The GTase associates with the phosphorylated
C-terminal domain of the largest subunit of pol II (CTD-P) (29, 30, 41,
43). Ceg1 by itself is inactive on CTD-P unless it is interacting with
the central region (amino acids 235-265) of Cet1 (10, 11). In
contrast, the GTase domain of MCE (MCE-(211-597)) is activated by
binding to CTD-P (44). The GTases from other fungi,
Schizosaccharomyces pombe (pce1), and Candida
albicans (Cgt1) do not require RTPase activation (45, 46).
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Fig. 4.
The isolated metazoan RTPase domain can
replace Cet1 in S. cerevisiae. A,
complementation by plasmid shuffling. YSB719 was transformed with
pDB20H-MCE-(211-597). A His+ isolate was subsequently
transformed with the following LEU2/2-µm plasmids: vector
(pRS425 (81)); CET1 (pAD5-CET1); CEL-1
(13-221) (pAD5-CEL-1-(13-221), expressing CEL-1-(13-221)
tagged with an HA epitope from the ADH1 promoter); MCE
(1-210) (pAD5-MCE-(1-210), expressing MCE-(1-210) tagged with
the HA epitope from the ADH1 promoter). Leu+
His+ transformants were tested for growth in the presence
of 5-FOA to shuffle out pRS316-CEG1-CET1. B, YSB719 was
transformed with pAD5-CEL-1-(13-221) (left plate) or
pAD5-MCE-(1-210) (right plate). Leu+ isolates
were subsequently transformed with the following HIS3/2-µm
plasmids: vector (pRS423) (81)), CEG1 (pRSH-CEG1),
MCE-(211-597) (pDB20H-MCE-(211-597)), CGT1
(pRSH-CGT1), pce1+ (pDB20H-pce1+),
or A103R (pDB20H-A103R, expressing the Chlorella virus GTase
A103R from the ADH1 promoter). Growth was tested after
plasmid shuffling. C, immunoprecipitation and GTase-GMP
intermediate formation assay. YSB719 transformants selected by 5-FOA
were grown in selective media, and whole-cell extracts were prepared.
Lane 1 has extract from YSB719 transformed with pAD5 and
pDB20H-pce1+. This extract was prepared from cells without
being selected by 5-FOA. Lanes 2 and 3 have
extracts from the same strain transformed with pAD5-CET1 and either
pRSH-CGT1 (lane 2) or pDB20H-pce1+ (lane
3), respectively. Lane 4 is from cells containing
pAD5-CEL-1-(13-221) and pDB20H-pce1+. Lane 5 is
from cells containing pAD5-CEL-1-(13-221) and pRSH-CGT1. Lane
6 is from cells containing pAD5-MCE-(1-210) and
pDB20H-pce1+. Lane 7 is from cells containing
pAD5-MCE-(1-210) and pRSH-CGT1. Immunoprecipitation was carried out
with 10 µg of extract protein, protein A-Sepharose beads, and
monoclonal antibody 12CA5. Precipitates were incubated for 15 min at
30 °C with 3 µM [ -32P]GTP and
processed to SDS-PAGE. Proteins were transferred to nitrocellulose
membrane and analyzed by immunoblotting with 12CA5 (upper
panel) and autoradiography to detect radiolabeled GTase-GMP
covalent intermediate (middle panel). The lower
panel shows the result of GTase-GMP formation with 20 µg of
whole-cell extract protein. The asterisks denote the
position of radioactive phosphate. The only combination that shows an
interaction is S. cerevisiae Cet1 and the C. albicans Cgt1 (lane 2).
cet1
cells
expressing pce1, Cgt1, or MCE-(211-597), the CEL-1 fragments 1-221,
13-221, or 1-585 could support viability (Fig. 4 and data not shown).
When overexpressed in ceg1
cet1
strain in
combination with MCE-(211-597), cells containing Cet1 formed colonies
after 1 day, whereas cells containing either CEL-1-(13-221) or
MCE-(1-210) formed colonies after 3 days (Fig. 4A). Both
S. pombe pce1 and C. albicans Cgt1 GTases could
also combine with the CEL-1 or MCE RTPase domains to support viability,
but the Chlorella virus GTase A103R (34) could not (Fig.
4B). A103R could not complement a ceg1
strain either even though enzyme-GMP complex was detectable in
lysates.3
-32P]GTP to detect any GTase (Fig. 4C,
middle panel). Lane 2 shows HA-Cet1
coprecipitates the C. albicans GTase Cgt1. In contrast, lane 3 shows that HA-Cet1 does not interact with the
S. pombe GTase pce1. These findings confirm our earlier
observation that pce1 functions without any interaction with RTPase
protein (46). Under the same conditions, neither pce1 (lanes
4 and 6) nor Cgt1 (lanes 5 and 7)
is coprecipitated with the RTPases CEL-1-(13-221) or MCE-(1-210).
Therefore, we conclude that the overexpressed metazoan RTPase domains
can function in vivo without linkage to the GTase domain.
strain because they do not bind and
activate Ceg1. To confirm this, regions of Cet1 were fused to the
RTPase domain of CEL-1-(13-221). The chimeras were co-expressed along
with Ceg1 overexpressed from a 2-µm plasmid in a
cet1
ceg1
strain (Fig. 5). Cet1-(1-225)-CEL-1-(13-221) cannot
functionally replace Cet1. In contrast, Cet1-(1-265)-CEL-1-(13-221),
which contains the Ceg1 interaction region (amino acids 235-265), can
support viability. When amino acids 1-265 are derived from the mutant
cet1-446 (P245A/W247A), which is disrupted for the ability to
interact with Ceg1 (11), the ability to replace Cet1 was disrupted. All
of the chimeric RTPases support viability of the same strain if
MCE-(211-597) replaces Ceg1 as the GTase (Fig. 5, right),
showing that these proteins are functional. These data support our
earlier conclusion that residues 235-265 of Cet1 are primarily
required for proper function of Ceg1.
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Fig. 5.
The addition of the
Ceg1-interacting region from Cet1 onto CEL-1 RTPase domain allows it to
function in the presence of Ceg1. YSB719 was transformed with
(left) pRSH-CEG1 or (right)
pDB20H-MCE-(211-597). His+ isolates were subsequently
transformed with the following LEU2/2 µm plasmids: vector
(pAD5); CET1 (pAD5-CET1);
CET1-(1-225)-CEL-1-(13-221) (pAD5-CET1
(1-225)-CEL-1-(13-221), expressing a fusion protein containing
residues 1-225 of Cet1 and CEL-1-(13-221) tagged with the HA
epitope); CET1-(1-265)-CEL-1-(13-221) (pAD5-CET1
(1-265)-CEL-1-(13-221));
cet1-446-(1-265)-CEL-1-(13-221)
(pAD5-cet1-446-(1-265)-CEL-1-(13-221), expressing a fusion protein
consisting of residues 1-265 from the cet1-446 mutant (11) and
CEL-1-(13-221) tagged with the HA epitope); and
CEL-1-(13-221) (pAD5-CEL-1-(13-221)). Leu+
His+ transformants were grown in the presence of 5-FOA, and
the plates were incubated for 3 days at 30 °C.
-32P]GTP-terminated RNA (Fig.
6B). C136S and R142K, mutated in key active site residues,
were inactive. The activities of E111Q, D112N, and D76N were about 20, 50, and 10%, respectively, that of the wild-type protein.
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Fig. 6.
RTPase activities of CEL-1-(13-221)
mutants. A, purification of recombinant
polyhistidine-tagged CEL-1-(13-221) protein. The peak fractions of the
heparin-Sepharose CL-6B column were analyzed by SDS-PAGE and visualized
by Coomassie Brilliant Blue staining (top panel) or
immunoblotting with monoclonal anti-polyhistidine antibody
(bottom panel). 400 and 50 ng of protein were loaded for the
top and bottom panels, respectively. Lane
1, wild-type CEL-1-(13-221); lane 2, C136S; lane
3, R142K; lane 4, D76N; lane 5, E111Q;
lane 6, D112N. For reasons that are not clear, the D76N
mutant shows slightly altered mobility. B, RTPase assay. The
wild-type and mutated CEL-1-(13-221) proteins were incubated at
30 °C for 10 min with 1 µM termini of a
[ -32P]GTP-labeled 65 nucleotide RNA. Reaction mixtures
were analyzed by thin layer chromatography (TLC) on
polyethyleneimine-cellulose plates. Released phosphate was detected by
autoradiography, and radioactive spots were cut out and quantitated by
liquid scintillation counting. Relative amounts of released phosphate
were plotted versus protein amount. C, in
vivo analysis of CEL-1-(13-221) mutants by plasmid shuffling.
YSB719 carrying pDB20H-MCE-(211-597) was transformed with the vector
pAD5 or derivatives expressing the indicated CEL-1 alleles.
Leu+ His+ transformants were tested for growth
in the presence of 5-FOA. Plates were incubated for 4 days at 30 °C.
D, immunoblot analysis of S. cerevisiae
whole-cell extracts. YSB719 cells carrying pDB20H-MCE-(211-597) and
CEL-1-(13-221) derivatives were grown in selective media but without
shuffling out the CEG1/CET1 plasmid. Extracts were prepared,
and 10-µg protein was analyzed by SDS-PAGE and immunoblotting with
the anti-HA antibody12CA5. Lane 1, pAD5; lane 2,
pAD5-CEL-1-(13-221); lane 3, pAD5-CEL-1-(13-221)C136S;
lane 4, pAD5-CEL-1-(13-221)C136A; lane 5,
pAD5-CEL-1-(13-221)R142K; lane 6,
pAD5-CEL-1-(13-221)R142A; lane 7, pAD5-CEL-1-(13-221)D76N;
lane 8, pAD5-CEL-1-(13-221)E111Q; lane 9,
pAD5-CEL-1-(13-221)D112N.
cet1
strain that was also expressing
MCE-(211-597) (Fig. 6C). CEL-1 mutants C136S, C136A, R142K,
or R142A could not support viability, whereas E111Q or D112N grew as
well as the wild-type strain. The in vivo phenotypes
correlated well with in vitro results. D76N, which had only
10% of wild-type activity in vitro, did not support
viability. Immunoblotting of whole-cell extracts confirmed that all the
mutants were expressed, although some variability in levels was
observed (Fig. 6D). The differences in ability to support
cell growth did not correlate with protein expression, since the
non-functional C136S, R142K, and R142A mutants were expressed at
greater levels than the functional E111Q and D112N proteins.
(pppApC; bold denotes the position
of the radioactive phosphate) or
(pppApC) positions
(Fig. 7B). CEL-1-(13-221) releases the terminal phosphate to leave a diphosphate end. This activity was not seen with
CEL-1-(13-221)C136S, mutated at the active site cysteine.
CEL-1-(13-221) did not have detectable nucleotide phosphohydrolase
activity under these conditions (Fig. 7C).
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Fig. 7.
Substrate specificity and catalytic
properties of the CEL-1 RTPase domain. A, substrates
for the RTPase assay. [ -32P]ATP-terminated
oligoribonucleotides (pppA(pC)npC,
n = 0-3) were synthesized with T7 DNA primase and
purified with DEAE-Sephadex A-25 column chromatography. Each RNA was
resolved by electrophoresis on a 36% polyacrylamide gel containing 3 M urea, and 32P label (asterisk) was
detected by PhosphorImager. Lanes 2-5, 80 pmol of termini
of dimeric (pppApC), trimeric (pppApCpC),
tetrameric (pppApCpCpC), and pentameric
(pppApCpCpCpC) RNAs, respectively. In lane 1,
[
-32P]ATP was loaded as a marker. B, RTPase
activity. Left panel, CEL-1-(13-221) releases the
-phosphate. The indicated amounts of the wild-type protein
(CEL-1-(13-221), lanes 3-5) and the active site cysteine
mutant (CEL-1-(13-221)C136S, lanes 6-8) were
incubated for 10 min at 30 °C with 0.1 µM
(termini) [
-32P]ATP-terminated dinucleotide RNA
(pppApC). The reaction mixtures were analyzed by TLC on
polyethyleneimine-cellulose plates, and 32P label
(asterisk) was detected by PhosphorImager. In lane
1 of each panel, the substrate was incubated with 1 unit of calf intestinal phosphatase (CIP) to determine the
position of free phosphate. Right panel, CEL-1-(13-221)
leaves a 5' diphosphate end. Reactions in lanes 1-8 were
identical to those in lanes 1-8 of the left
panel, except [
-32P]ATP-terminated dinucleotide
RNAs were replaced with [
-32P]ATP-terminated
dinucleotide RNA (pppApC). The position of ppApC was
determined using ADP-terminated dimer as standards (8). C,
nucleotide phosphohydrolase activity. Reactions in lanes
1-5 were identical to those in lanes 1-5 in the
left panel of B, except
[
-32P]ATP-terminated dinucleotide RNAs were replaced
with 5 µM [
-32P]ATP (pppA).
Incubation was for 30 min at 30 °C.
-
phosphodiester bond of trinucleotide more efficiently than that
of dinucleotide. Little difference was seen between tri-, tetra-, and
pentanucleotides. With a double-reciprocal plot, the
kcat/Km values with ATP,
dinucleotide, trinucleotide, and tetranucleotide were calculated to be
5.5 × 10, 0.5 × 105, 4.6 × 105, 5.1 × 105
M
1 s
1, respectively. The same
length dependence was observed with full-length MCE and MCE-(1-210)
(data not shown), indicating that two phosphodiester bonds are
necessary for optimal fit of the RNA substrate into the active site of
metazoan capping enzyme RTPases.
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Fig. 8.
Effect of RNA chain length on the CEL-1
RTPase. The indicated amounts of CEL-1-(13-221) were incubated
for 10 min at 30 °C with 1 µM termini of
[ -32P]ATP-terminated di-, tri-, tetra-, and
pentanucleotides. The reaction mixtures were analyzed by TLC on
polyethyleneimine-cellulose plates. 32P label was detected
by autoradiography, and radioactive spots on
polyethyleneimine-cellulose plate were cut out and counted by liquid
scintillation.
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Fig. 9.
Cel-1 is required for embryonic development
and CTD serine 2 phosphorylation. A, terminal
developmental arrest of cel-1(RNAi) embryos.
Wild-type (WT) and RNAi embryos were examined by
differential interference (DIC) microscopy. The wild-type
embryo in the left panel is about to hatch, but
ama-1(RNAi) (pol II) and cel-1(RNAi) embryos each
arrested with ~100 cells. Embryos measure ~50 µm. B,
expression of RNA pol II and the CDK-9 kinase in wild-type and
cel-1(RNAi) embryos. Embryos were stained simultaneously
with 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) to
visualize DNA, a CDK-9 antibody (23), and an antibody to the
unphosphorylated pol II large subunit (8WG16). C, CTD serine
5 phosphorylation in cel-1(RNAi) embryos.
Wild-type or RNAi embryos (presented in rows) were stained
with 4',6'-diamidino-2-phenylindole hydrochloride, an
serine/arginine-rich protein antibody (16M3) as a control, or an
antibody to CTD phosphoserine 5 (26), as indicated. An expanded
-Ser(P)-5 stained somatic nucleus (white arrow) is shown
in the right column. Representative embryos at comparable
stages are presented. D, cel-1 is required for
CTD serine 2 phosphorylation. Wild type or RNAi embryos were stained
with 4',6'-diamidino-2-phenylindole hydrochloride, a C/EBP-binding
protein (CBP)-1 antibody for a staining control (82), and an antibody
to CTD phosphoserine 2 (H5) (83, 84). An expanded
-Ser(P)-2-stained
somatic nucleus is shown as in C. In the transcriptionally
silent germline precursor (red arrow), only weak
cross-reactivity with perinuclear germline P granules is detected.
Mitotic nuclei, which also cross-react with
-Ser(P)-2 in the absence
of the pol II epitope (23, 63), are marked with asterisks.
Representative embryos of comparable stages are shown.
-Ser(P)-5 and
-Ser(P)-2 for clarity (Fig. 9, C and
D).
-Ser(P)-5 and
-Ser(P)-2 staining depends upon transcription.
Staining with
-Ser(P)-5 and
-Ser(P)-2 is not detected in
embryonic nuclei until the three-to-four cell stage, when new mRNA
transcription begins (63). At later stages, the patterns and intensity
of this staining closely parallel transcription activity in embryonic cells. For example, both types of staining are eliminated or reduced in
tandem by RNAi depletion of transcription initiation factors such as
TFIIB (ttb-1) (24, 27). In contrast, when the elongation factor CDK-9 is depleted by RNAi, Ser-5 phosphorylation levels appear
normal, but Ser-2 phosphorylation is undetectable (23).
-Ser(P)-2 staining were dramatically reduced, to a level only slightly higher than the background observed in ama-1(RNAi) embryos (Fig. 9D).
Levels of the CTD Ser-2 kinase CDK-9 appeared to be normal in
cel-1(RNAi) embryonic nuclei, arguing that the
drop in CTD phosphorylation was not an indirect effect (Fig.
9B). The specific and substantial defect in CTD Ser-2
phosphorylation suggests that when CEL-1 levels are depleted, the
normal progression of CTD phosphorylation during transcription is
disrupted at most or possibly all genes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Fig. 3). Therefore, CEL-1 residues 586-623
are not required for protein stability or proper localization but are
essential for GTase activity.
-phosphate from
the 5' end of a dinucleotide (Fig. 7 and data not shown). However,
maximal activity is observed on substrates that are three nucleotides
or longer (Fig. 8 and data not shown). The S. cerevisiae RTPase Cet1 is unrelated to PTPs, and its reaction mechanism is different from that of metazoan RTPases (40). However, Cet1 also acts
on dinucleotide and trinucleotide RNAs efficiently (18).3
Diphosphate-ended oligonucleotides such as ppApG, ppGpC, and ppGpCpC
are active as guanylyl acceptors for mammalian and yeast GTases
(68-72). Structural studies on RNA polymerase II suggest that RNA
exits polymerase in the vicinity of the CTD (73), where capping enzymes
will be bound. Capping occurs around the time mRNAs are about 30 nucleotides in length (74, 75). Therefore, capping enzyme probably
recognizes the first few phosphodiester bonds of nascent RNA that
emerge from the body of pol II and immediately caps the mRNA.
strain (52). Transfection experiments
showed that MCE-(1-210) is mostly cytoplasmic in mammalian cells (51).
This may also be true in S. cerevisiae. Overexpression may
be necessary to drive sufficient amounts of RTPase into the nucleus and
into proximity with the mRNA 5' end. Alternatively, RTPases may
independently bind pol II or a pol II-associated protein. HIV-1 Tat
protein binds to both full-length MCE as well as the isolated GTase and
RTPase domains (77). There could be a corresponding cellular protein(s)
that mediates the association of RTPase domain with the pol II complex
or RNA chain. Whatever mechanism is used, isolated RTPase domains
function more efficiently in vivo when it is linked to a
GTase domain.
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ACKNOWLEDGEMENTS |
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We thank Drs. Aaron J. Shatkin and Fabio Piano for communicating results before publication, Gary Ruvkun for supplying C. elegans nuclear extract, Dale Wigley for pET-A103R, Gerhard Wagner and Kylie Walter for pT7-911Q, Takahiro Kusakabe for designing the sequences of synthetic oligonucleotides for preparation of short RNA with T7 DNA primase, and Robin Buratowski for help with the plasmid and oligo tables.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM56663 (to S. B.) and GM62891 (to T. K. B.).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.
§ Senior Postdoctoral Fellow of the American Cancer Society, Massachusetts Division. Present address: Dept. of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454.
Postdoctoral Fellow For Research Abroad of the Japan Society
for the Promotion of Science.
** Present address: Mayo Clinic, Rochester, MN 55901.
Present address: Laboratory of Seeds Finding Technology, Eisai
Co., Ltd., Ibaraki 300-2635, Japan.
§§ A scholar of the Leukemia And Lymphoma Society. To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0696; Fax: 617-738-0516; E-mail: steveb@hms.harvard.edu.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M212101200
2 T. Takagi, A. K. Walker, C. Sawa, F. Diehn, Y. Takase, T. K. Blackwell, and S. Buratowski, unpublished information.
3 T. Takagi, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: RTPase, RNA 5'-triphosphatase; GTase, GTP::mRNA guanylyltransferase; PTP, protein-tyrosine phosphatase; RNAi, RNA interference; 5-FOA, 5-fluoroorotic acid; pol II, RNA polymerase II; CTD, C-terminal domain of the largest subunit of RNA polymerase II; CTD-P, phosphorylated CTD; HA, influenza virus hemagglutinin; EST, Expressed Sequence Tag; ORF, open reading frame; P-TEFb, positive transcription elongation factor b; CDK, cyclin-dependent kinase; kb, kilobase; DTT, dithiothreitol; ds, double-stranded; MCE, mouse capping enzyme; BVP, baculoviral PTP.
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