mRNA Capping Enzyme Requirement for Caenorhabditis elegans Viability*

Priya SrinivasanDagger §, Fabio Piano, and Aaron J. ShatkinDagger §||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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.

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 epsilon -amino group of the conserved lysine (2, 3).

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).

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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).

RNA 5' Triphosphatase Assay-- T7 polymerase run-off RNA (72-mer) labeled with [gamma -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.

Capping Enzyme Guanylylation and RNA Cap Formation-- In vitro translated and affinity-purified proteins bound to beads were incubated in buffer B with [alpha -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.

For cap formation T7 polymerase run-off RNA (72-mer), synthesized as above with GTP instead of [gamma -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).

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 Delta  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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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 Delta ) 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).

C. elegans RNA Triphosphatase Activity-- In vitro synthesized, affinity-purified GST fusion proteins were tested for TPase activity using a [gamma -32P]GTP 5' end-labeled RNA 72-mer as substrate. As shown by TLC analysis (Fig. 3), protein encoded by the Delta  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 [gamma -32P]GTP by calf intestinal alkaline phosphatase (lane 1). In contrast to clone Delta , 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 [gamma -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.

RNA Guanylyltransferase Activity of C. elegans Capping Enzyme-- The expressed products were also tested for guanylyltransferase activity using [alpha -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 [alpha -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.

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 [alpha -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 [alpha -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).

C. elegans Capping Enzyme with Extended Termini Complements S. cerevisiae Delta CEG1 and Delta 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 (Delta CEG1, Fig. 6) or TPase (Delta CET1). The C. elegans capping enzyme constructs were cloned into plasmids containing S. cerevisiaeTRP1 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 Delta Cet1 (Fig. 6). However, it did support the growth of Delta 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 Delta Ceg1 strain. It also failed in Delta 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 (Delta CEG1) or triphosphatase (Delta 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 Delta  (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).

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 Delta CEG1 and Delta CET1 strains, indicating that initiation at the first methionine in the N + C construct is sufficient for capping function.

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).


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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.

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: TPase, RNA triphosphatase; GTase, RNA guanylyltransferase; RNAi, RNA interference; ds, double-stranded; GST, glutathione S-transferase.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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