From the Center for Advanced Biotechnology and
Medicine and § Graduate Program in Biochemistry,
Graduate School of Biomedical Sciences, University of Medicine and
Dentistry of New Jersey, Piscataway, New Jersey 08854 and
¶ Department of Biology, New York University,
New York, New York 10012
Received for publication, November 27, 2002
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ABSTRACT |
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Capping of the initiated 5' ends of RNA
polymerase II products is evolutionarily and functionally conserved
from yeasts to humans. The m7GpppN cap promotes RNA
stability, processing, transport, and translation. Deletion of capping
enzymes in yeasts was shown to be lethal due to rapid exonucleolytic
degradation of uncapped transcripts or failure of capped but
unmethylated RNA to initiate protein synthesis. Using RNA interference
and Caenorhabditis elegans we have found that RNA capping
is also essential for metazoan viability. C. elegans
bifunctional capping enzyme was cloned, and capping activity by the
expressed protein as well as growth complementation of yeast deletion
strains missing either RNA triphosphatase or guanylyltransferase required terminal sequences not present in the previously isolated cel-1 clone. By RNA interference analysis we show that
cel-1 is required for embryogenesis.
cel-1(RNAi) embryos formed cytoplasmic granules
characteristic of a phenocluster of RNA processing genes and died early
in development.
Eukaryotic mRNAs are modified at the 5' end by
addition of a m7GpppN "cap" (1). This characteristic
structure is conserved among cellular and most viral gene transcripts,
in many cases synthesized by different RNA polymerases (2, 3). Caps are produced co-transcriptionally on RNA polymerase II nascent transcripts via three enzymatic reactions: (i) RNA triphosphatase
(TPase)1 removes the
Plants and metazoans contain bifunctional capping enzymes consisting of
N-terminal TPase and C-terminal GTase domains, whereas the RNA
methyltransferase is a separate protein (2, 3). By contrast, in yeast
species the three reactions for cap synthesis are each catalyzed by a
separate enzyme (5-7). The TPase domain of metazoan capping enzymes
includes a conserved active site motif, (I/V)HCXXGXXR(S/T)G, which is also
characteristic of protein tyrosine phosphatases (8, 9).
These TPases, like the protein phosphatases, form a
cysteinyl-phosphate intermediate as part of their catalytic mechanism
(8-11). TPases of unicellular eukaryotes do not contain the Cys motif
and use a different catalytic mechanism (7, 12). GTases of metazoan and
unicellular origin share several identifiable motifs characteristic of
a nucleotidyltransferase superfamily (13), including a highly conserved
KXDG sequence. This motif, and notably the lysine, is
necessary for formation of an enzyme-guanylylate GTase intermediate via
phosphoamide linkage of GMP to the In yeast species loss of any one of the three cap-forming enzymes is
lethal (5-7). Despite differences in sequence, structural organization, and TPase catalytic mechanism, mammalian capping enzyme
cDNA clones effectively complement growth of yeast deletion strains, demonstrating evolutionary and functional conservation of
capping from unicellular to higher eukaryotes (14, 15). What has not
been tested previously is whether RNA capping is also essential for
metazoan viability. For this purpose we used RNA interference (RNAi) in
Caenorhabditis elegans (16). Based on sequence comparisons,
a C. elegans putative bifunctional capping enzyme gene
(cel-1) was identified previously, and the
bacterially expressed N-terminal domain was shown to encode functional
TPase (8). However, GTase was not obtained either as a C-terminal domain or bifunctional capping enzyme (8). We have cloned enzymatically active C. elegans bifunctional capping enzyme containing
additional N- and C-terminal sequences. They include a 3' exon not
obtained in the previously reported, presumptive full-length clone but which we found is necessary for TPase, GTase, and RNA capping activity
as well as for complementation of yeast deletion strains. Double-stranded (ds) RNA corresponding to capping enzyme fed or injected into C. elegans was shown to induce high penetrance
embryonic lethality. Progeny of RNAi-treated animals died early in
development, showing characteristics of the RNAi phenotypes of other
RNA processing genes (17).
Cloning of Capping Enzyme cDNAs--
The C. elegans capping enzyme sequence was identified on cosmid C03D6
(Wormpep C03D6.3). ESTs yk798b08, yk786d02, yk784d09, and yk790d02
corresponding to capping enzyme were provided by Dr. Yuji Kohara
(National Institute of Genetics, Mishima, Japan). Reverse
transcription-PCR (Invitrogen) was used to isolate a full-length capping enzyme clone with C. elegans total RNA as template
and gene-specific 5' and 3' primers based on the EST sequences
5' (5'-GCGCGGATCCATGGCTACACGAGGACCAACACCTGACAAAGCG-3') and 3'
(5'-GCGCGGATCCAAATGTAGGAGAATGATTATCAGAGTTTTTGTC-3'). A new capping
enzyme clone that initiated at the thirteenth amino acid downstream of
the first methionine was also obtained using as 5' primer
(5'-GCGGATCCATGGGACTGCCTGATAGATGGCTGCA-3') and the same 3'
primer. Subclones were generated by PCR from the full-length cDNA.
The cDNAs with BamH1 sites at the 5' and 3' ends were
cloned into pCR2.1 (Invitrogen) and sequenced to ensure that no
mutations were introduced during subcloning.
Plasmids--
For protein expression, all C. elegans
capping enzyme constructs generated by PCR were subcloned from pCR 2.1 into the BamH1 site of pET42a (Novagen), in-frame with the
N-terminal glutathione S-transferase (GST) tag. Mutagenesis
at the enzyme active sites was performed using the QuikChange
site-directed mutagenesis kit (Stratagene) and appropriate primers. All
mutations were verified by sequencing.
For S. cerevisiae complementation, constructs were subcloned
into the BamH1 site of yeast shuttle vectors p424
(2µ TRP1) or p414 (CEN TRP1) (18).
Expression of the C. elegans genes in these plasmids is
under the control of the constitutive yeast glyceraldehyde-3-phosphate
dehydrogenase promoter. All constructs were verified by sequencing.
Protein Expression and Purification--
pET42a plasmids
containing the different capping enzyme clones were used to express GST
fusion proteins in the TNT T7 Quick Coupled Reticulocyte Lysate System
(Promega). Products were affinity-purified by binding to GST-agarose
beads (Sigma), which were then washed extensively in buffer A (50 mM Tris, pH 7.5, 100 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM RNA 5' Triphosphatase Assay--
T7 polymerase run-off RNA
(72-mer) labeled with [ Capping Enzyme Guanylylation and RNA Cap Formation--
In
vitro translated and affinity-purified proteins bound to beads
were incubated in buffer B with [
For cap formation T7 polymerase run-off RNA (72-mer), synthesized as
above with GTP instead of [ Complementation of S. cerevisiae Deletion Mutants by C. elegans
Capping Enzyme Clones--
Haploid S. cerevisiae strains
YBS2 [MATa, leu2, lys2,
trp1, ceg1::hisG,
p360-(CEN, URA, CEG1)] and YBS20
[MATa, trp1, his3, ura3,
leu2, ade2, can1,
cet1::LEU2,
p360-(CEN,TRP,CET1)] (11, 13) were transformed with
C. elegans capping enzyme clones in either multi-copy 2µ
or single-copy CEN expression plasmids using lithium acetate.
Trp+ transformants obtained at 30 °C were tested for the
ability to grow in the presence of 5-fluoroorotic acid (FOA), which
selects against cells that retain either the CEG1- or
CET1-containing URA3 plasmid (21).
RNAi Methods--
dsRNA was introduced into C. elegans N2 (Bristol) strain using either "feeding" from
bacterial expression (22) or injection of dsRNA synthesized in
vitro from the Phenotypic Analysis--
Time-lapse digital movies were made
essentially as previously described (23). Embryos were dissected out of
animals at ~36 h after injection of dsRNA and mounted on a 2%
agarose pad. Time-lapse analyses of early embryos were made using a
Leica DMLA microscope with a 40× lens and differential
interference contrast optics to record 300 frames at 10-s intervals.
Terminal phenotypes were recorded from 18-22-h-old embryos that had
been incubated at 25 °C (wild-type embryos hatch after 12 h at
this temperature) using a Hamamatsu Orca II camera mounted on a Leica
DMRA2 microscope and a 63× lens and differential interference contrast
optics. Openlab software (Improvision) was employed for the digital capturing.
Identification and Cloning of C. elegans mRNA Capping
Enzyme--
Previous sequence analyses (8) indicated that C. elegans contains a bifunctional capping enzyme with conserved
active sites and other defining motifs and significant sequence
similarity to other capping enzymes (Fig.
1A). C. elegans
TPase expressed in bacteria required for divalent cation-independent
enzymatic activity a cysteine present at position 124 in an
active site motif. This same mechanism was also
demonstrated for mammalian TPase (10, 11) and is completely different
from the metal cofactor-dependent TPase of S. cerevisiae and other unicellular organisms (12). In the previous
studies (8), TPase was obtained as an active, soluble protein in
Escherichia coli transformed with the N-terminal domain of
the C. elegans capping enzyme clone (cel-1).
However, GTase could not be expressed in either bacteria or
reticulocyte lysate. Sequence comparisons did not reveal any other
putative capping enzymes in C. elegans, but recent EST data from a large scale cDNA
project2 and from an ovary
cDNA library (23) suggest that the C. elegans capping
enzyme contains additional terminal sequences. These analyses also
suggested that Wormpep C03D6.2 is part of cel-1 (C03D6.3). These new, additional sequences could be required for GTase activity. Based on this information, reverse transcription-PCR was used to
isolate capping enzyme cDNA clones encoding proteins with extended N and C termini compared with cel-1 (Fig.
1B).
In Vitro Expression of C. elegans Capping Enzyme
Clones--
The previously studied (8) 574-amino acid open
reading frame clone (cel-1, referred to here as clone C. elegans RNA Triphosphatase Activity--
In vitro
synthesized, affinity-purified GST fusion proteins were tested for
TPase activity using a [ RNA Guanylyltransferase Activity of C. elegans Capping
Enzyme--
The expressed products were also tested for
guanylyltransferase activity using [ RNA Capping Activity of C. elegans Bifunctional
Enzyme--
To verify that the results obtained with separate TPase
and GTase also apply to coupled reactions that result in caps, RNA was
incubated with [ C. elegans Capping Enzyme with Extended Termini
Complements S. cerevisiae
The N + C clone contains a potential initiator codon at amino acid 13 in-frame with the putative start codon at position one. The possibility
that synthesis of the capping enzyme begins at the downstream ATG in N + C was tested by mutating the methionine codon at position 13 to
alanine (N + C M13A). The resulting clone complemented growth in both
RNA Interference by C. elegans Capping Enzyme
Double-stranded RNA--
The combined findings indicate that C. elegans bifunctional capping enzyme is encoded in 13 exons and
contains a total of 623 amino acids. To test if capping enzyme is
essential for the viability of a metazoan, dsRNA corresponding to
nucleotides 1-1722 (encoding amino acids 1-574) of the worm,
bifunctional protein was injected into C. elegans (23). The
C. elegans strain used in these experiments showed less than
1% embryonic lethality when treated with dsRNA from more than 350 genes tested as part of a large RNAi project (17). However, when
treated with dsRNA from the capping enzyme gene (cel-1),
100% of the embryos failed to hatch (Fig.
7). One- or two-cell
cel-1(RNAi) embryos contained cytoplasmic
granules not usually seen in wild-type embryos. These granules,
however, are commonly observed when other genes predicted to function
in mRNA processing are tested (17). Despite the presence of
aberrant cytoplasmic granules, other basic cell biological events
appeared normal during the first embryonic cell divisions, consistent
with the idea that these early stages are not dependent on newly
transcribed genes (27). However, analysis of terminal phenotypes showed
defects that included embryos with no gut granules (94%,
n = 79) but variable levels of differentiation
including twitching (a sign of muscle differentiation) and other cell
types. Similar results were obtained by feeding dsRNA (data not
shown).
A C. elegans putative cap methyltransferase clone C25A1.3
(1149 nucleotides) coding for a 383-amino acid protein was also isolated based on sequence alignments and data bank comparisons. Translation products directed by the clone had low enzymatic activity (<1% of recombinant human RNA methyltransferase) and did not
complement a yeast ABD1 deletion strain (data not shown).
C. elegans treated with duplex RNA corresponding to the
putative full-length RNA methyltransferase clone elicited an embryonic
lethal phenotype with low penetrance (9%, n = 384).
mRNA capping is characteristic of eukaryotic
cells. Caps are added to RNA polymerase II nascent transcripts by the
sequential action of TPase, GTase, and RNA methyltransferase. These
enzymes have been cloned from mammalian, yeast, and other cellular and viral origins, and their study has facilitated insights into extensive biochemical linkages, for example between transcription and RNA processing, including capping, that regulate gene expression (4, 28,
29). In yeasts and other unicellular species the three cap-forming
reactions are catalyzed by separately encoded proteins, and the
corresponding genes are necessary for viability. In multicellular organisms capping occurs by the same series of reactions, but they are
carried out by only two proteins, bifunctional capping enzyme with
TPase and GTase in the N- and C-terminal domains, respectively, and a
separately encoded RNA methyltransferase. In addition, metazoan and
unicellular TPases use very different catalytic mechanisms. Despite
these variations on the basic theme, mammalian capping enzymes
complement growth of yeast deletion strains, consistent with
evolutionary conservation of a process with important functional
consequences at multiple levels of gene expression including initiation
of transcription, splicing, nucleocytoplasmic transport, translation,
and RNA turnover.
RNAi provided an opportunity to test the effects of depleting capping
enzyme in a multicellular organism. To assure that the authentic RNA
was used, C. elegans capping enzyme clones were isolated,
and expressed proteins were analyzed for TPase, GTase, and RNA capping
activities. Previously, bacterial expression of the N-terminal portion
of a putative bifunctional capping enzyme clone demonstrated that it
encoded TPase (8). However, despite sequence similarities to other
capping enzymes, cloning and expression of C. elegans GTase
remained elusive. The results reported here provide the explanation,
namely that an additional, 13th exon encoding 37 C-terminal amino acids
is required. This extension is necessary for enzymatically active GTase
expressed either as the bifunctional protein or separately from a 3'
domain subclone. In our studies of the bifunctional protein, active
TPase was also obtained with the clone containing the additional 3'
exon, either with or without a 5' extension that consisted of another
ATG followed by 12 additional codons. Although the 5' domain previously
expressed in bacteria yielded active enzyme (8), TPase made in
vitro with this subcloned domain was inactive without the
N-terminal extension. Surprisingly, the C-terminal extension was
sufficient in the cell-free translated bifunctional protein not only
for GTase but also for TPase activity and RNA capping, suggesting that
the C-terminal sequences may facilitate overall protein folding into
domains that affect both enzymatic activities. Structural studies
should help elucidate the functional effects of the 3' exon sequences.
In contrast to the results with the in vitro products, growth complementation of yeast deletion strains was obtained only with
expression constructs, either single or multi-copy plasmids, which
encoded a 623-amino acid bifunctional protein containing both the N-
and C-terminal extensions. The N-terminal sequences may have a role in
protein-protein interactions that increases stability and/or promotes
capping in the context of the intact cell.
RNAi analysis using the full-length product of the capping enzyme
gene confirmed and extended initial observations made during a
large-scale RNAi project (23), including the presence of aberrant cytoplasmic granules in one-cell embryos. Recently, a new large-scale project identified a number of additional genes whose RNAi phenotype showed similar granules. Significantly, all the genes in this phenotypic class (phenocluster) whose putative function could be
assigned based on sequence analysis were predicted to play a role in
RNA processing (17).
In the present study, we found that cel-1(RNAi)
terminal phenotypes show variable amounts of differentiation but no gut
formation (as assayed by birefringent gut granules). These late
embryonic phenotypes are reminiscent of developmental mutations in
genes that are necessary to pattern the embryo properly (for example, see Kemphues et al. (30)). Several non-exclusive hypotheses can account for these findings. For example, the RNAi effect may be
variable, or cel-1 may have an indirect effect by altering the stability of some critical developmental genes. Another possibility is that cel-1 is differentially required in different
tissues by directly affecting post-transcriptional regulation of
specific developmental genes. We do not favor the first hypothesis
because we saw a robust RNAi effect (100% of the embryos from mothers treated with cel-1 dsRNA died). Future work will be required
to test if any specific connection exists between embryonic patterning and RNA capping in animals.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-phosphate from the initiated RNA 5' end, (ii) RNA guanylyltransferase (GTase) adds GMP from GTP to the resulting diphosphate terminus, and (iii) RNA (guanine-7) methyltransferase methylates the added guanosine. These early events in gene
expression protect pre-mRNAs from exonucleolytic attack and,
facilitated by cap-binding proteins (4), promote processing, transport of mature mRNAs to the cytoplasm, and translation initiation.
-amino group of the conserved
lysine (2, 3).
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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-mercaptoethanol, 20% glycerol) followed by buffer B
(25 mM Tris, pH 7.5, 5 mM MgCl2, 0.5 mM dithiothreitol) 3 times before performing enzyme
assays with the bound proteins. Soluble His6-tagged
full-length human capping enzyme was expressed from pHis (T)-hCAP1a
(19) as described previously (20).
-32P]GTP (50 µCi, 5000 Ci/mmol, Amersham Biosciences) was transcribed from BamH1-linearized pBlueScript SKII+ (Stratagene). The 5'
end-labeled RNA was passed through a Chroma Spin-10 column (BD
Biosciences) and incubated with translated, affinity-purified proteins
in buffer B. Products were analyzed by polyethyleneimine-cellulose thin layer chromatography (TLC) in 0.8 M acetic acid containing
0.9 M LiCl followed by autoradiography.
-32P]GTP (10 µCi,
3000 Ci/mmol, Amersham Biosciences) and 0.10 µg of inorganic
pyrophosphatase (Roche Molecular Biochemicals). Guanylylation of
proteins was analyzed by SDS-PAGE followed by autoradiography.
-32P]GTP, was incubated
with the different proteins in GTP-labeling buffer for 30 min at
25 °C. Cap formation was analyzed as described previously (14).
clone (see Fig. 2A) as previously
described (17). In preliminary tests, injection of dsRNA was found to
be more robust than "feeding" and, therefore, was used for the
phenotypic analysis.
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ABSTRACT
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RESULTS
DISCUSSION
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Fig. 1.
Capping enzyme sequence and
organization. A, sequence alignment of mRNA capping
enzymes from C. elegans, human, and
Drosophila. Amino acids in bold indicate the
active site motifs for the triphosphatase (N-terminal) and
guanylyltransferase (C-terminal) domains. Asterisks indicate
regions of complete identity, semicolons indicate conserved
amino acids, and dots indicate semi-conserved residues.
B, gene organization. i, the previously reported
(8) cel-1 clone C03D6.3 containing 12 exons.
ii, N + C clone encoding active CE contains 13 exons and
a 13-amino acid extension at the N terminus.
)
was extended either at the C terminus by 37 amino acids (+C), at the N
terminus by 12 residues (+N), or at both ends (N + C). Also constructed
were capping enzyme subclones for TPase expression either with (R + N)
or without (R) the N-terminal extension and for GTase with (G + C) or
without (G) the thirteenth exon (Fig.
2A). Although bacterial
expression of these constructs as GST, maltose binding protein, or
His6-tagged fusions yielded either insoluble aggregates or
very low levels of expression, soluble GST fusion proteins of the
predicted sizes were obtained in a coupled in vitro T7 transcription/translation system (Fig. 2B).
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Fig. 2.
C. elegans capping enzyme.
A, schematic representation of cDNA clones.
B, analysis of expressed proteins. Capping enzyme-GST fusion
proteins were synthesized in a coupled in vitro system, and
the [35S]methionine-labeled products were
affinity-purified using GST-agarose beads and analyzed by SDS-PAGE and
autoradiography. aa, amino acid(s).
-32P]GTP 5' end-labeled RNA
72-mer as substrate. As shown by TLC analysis (Fig.
3), protein encoded by the
clone
without extended termini (lane 5), like GST (lane
3) and products made without adding DNA (lane 4), had
no detectable TPase activity as compared with release of
32Pi by treatment with recombinant human
capping enzyme (lane 2) or of 32P from
[
-32P]GTP by calf intestinal alkaline phosphatase
(lane 1). In contrast to clone
, products directed by
clones that contained extensions at the N (lane 8) or C
termini (lanes 7 and 11) or both ends
(lanes 6 and 13) actively liberated
32Pi from the RNA substrate. TPase produced
from the subclone with the N-terminal extension was active (R + N,
lane 9) but was inactive without this additional sequence
(R, lane 10). Mutation of the active site cysteine to
serine, i.e. C124S in +C (lane 12) and C136S in
N+C (lane 15) nearly abolished TPase activity.
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Fig. 3.
RNA 5' triphosphatase activity. T7
polymerase run-off transcripts containing 5' terminal
[ -32P]GTP were incubated with the indicated in
vitro synthesized, affinity-purified C. elegans GST
fusion proteins or with bacterially expressed, purified human capping
enzyme (CE). GST and calf intestinal alkaline phosphatase
(CIP) were included as negative and positive controls,
respectively. Release of radiolabeled Pi was detected by
TLC and autoradiography.
-32P]GTP and
assaying the formation of radiolabeled capping intermediate Gp-GTase. Products without the 37-amino acid C-terminal
extension were inactive (Fig. 4,
lanes 4, 7, 11, and 12).
The addition of the new 3' exon predicted in Wormbase yielded active
bifunctional capping enzyme in the presence (lane 5) or
absence of the extended N terminus (lane 6). Consistent with
a requirement for the added 3'-terminal residues, active GTase was also
expressed from subclones with (lane 13) but not without them
(lane 12). Mutation of the active site lysine to alanine in
the KXDG motif (K311A in clone N + C, lane 8, and
K299A in clone +C, lane 9) abolished GTase activity.
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Fig. 4.
RNA guanylyltransferase activity. The
indicated in vitro translated, affinity-purified GST-tagged
C. elegans proteins were incubated with
[ -32P]GTP, and guanylylated, radiolabeled products
were analyzed by SDS-PAGE and autoradiography. Purified GST and
recombinant human capping enzyme (CE) were included as
controls.
-32P]GTP and the C-terminally extended
products either with (Fig. 5, lane
3) or without the extra N-terminal sequences (lane 5). In both cases the RNA triphosphate 5' end was converted to GpppG (Fig.
5). Cap structures were not formed when the C-terminal extension was
missing (lanes 2 and 4). These results provide
evidence for C. elegans cDNA clones encoding
bifunctional capping enzyme and indicate that capping activity in
vitro depends on the presence of the new 3' exon but not the
additional N-terminal sequences.
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Fig. 5.
mRNA cap formation. The indicated
in vitro translated and affinity-purified C. elegans GST fusion proteins or purified recombinant human capping
enzyme (CE) were incubated with T7 run-off transcripts and
[ -32P]GTP. Purified GST was used as a negative
control. Cap formation on the ~72-mer RNAs was verified by digestion
with P1 nuclease and CIP followed by TLC analysis with authentic GpppG
marker (Ambion).
CEG1 and
CET1 Deletion
Strains--
Functional conservation of capping enzymes has been
demonstrated previously by growth complementation of yeast deletion
strains transformed with mouse or human capping enzyme expression
constructs (14, 15). A plasmid shuffle strategy was used to determine whether C. elegans capping enzyme also could complement
haploid S. cerevisiae deletion strains that are missing
either GTase (
CEG1, Fig. 6)
or TPase (
CET1). The C. elegans capping enzyme
constructs were cloned into plasmids containing S. cerevisiae 2µ TRP1 or CEN TRP1 under
transcriptional control of the constitutive yeast glyceraldehyde
3-phosphate dehydrogenase promoter. In contrast to the in
vitro results, only constructs containing extensions at both
termini supported growth of the deletion strains. This same requirement
was seen with both single- and multicopy expression constructs (Fig. 6
and data not shown). Mutant clone N + C C136S, which encoded
essentially inactive TPase (Fig. 3, lane 15), failed to
complement
Cet1 (Fig. 6). However, it did support the
growth of
Ceg1, but to a lesser extent than the wild-type
N + C clone, consistent with a weak interaction of CET1 with mammalian
GTase (24). The inactive GTase mutant (N + C K311A) did not complement in the
Ceg1 strain. It also failed in
Cet1,
in agreement with CEG1 requiring CET1 for allosteric (25) and/or
stabilizing (26) interactions.
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Fig. 6.
Yeast growth complementation by C. elegans capping enzyme. Yeast strains lacking either
guanylyltransferase ( CEG1) or triphosphatase
(
CET1) were transformed with the indicated C. elegans capping enzyme expression plasmids. Trp+
FOA-resistant cells were selected for growth as described under
"Materials and Methods." Sectors contain cells transformed with
(1), N + C (2), +C (3), N + C K311A
(4), N + C C136S (5), N + C M13A (6), and
vector (7) or not transformed (8).
CEG1 and
CET1 strains, indicating that
initiation at the first methionine in the N + C construct is sufficient
for capping function.
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Fig. 7.
cel-1(RNAi)
embryonic phenotypes. Wild-type embryos at ~ 4 h
(a) and ~11 h (b) after fertilization (~2 h
before hatching). Panels c-f show terminal phenotypes
of ~ 20 h old cel-1(RNAi) embryos
that arrest as a mass of cells with various levels of
differentiation.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Y. Kohara for the EST clones, K. Mizumoto for pHis (T)-hCAP1a, S. Shuman for the yeast deletion strains, and M. Hampsey for the yeast plasmids. Drs. J. Mullen and S. Brill provided helpful advice on the complementation assays.
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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: Center for
Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5311; Fax: 732-235-5318; E-mail:
shatkin@cabm.rutgers.edu.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M212102200
2 Y. Kohara, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: TPase, RNA triphosphatase; GTase, RNA guanylyltransferase; RNAi, RNA interference; ds, double-stranded; GST, glutathione S-transferase.
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