The Caenorhabditis elegans mRNA 5’-capping enzyme: In vitro and in vivo characterization*

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 amino (N)-terminal region with RTPase activity and a carboxy (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 (PTP), so mutagenesis of residues predicted to be important for RTPase activity were carried out. CEL-1 uses a mechanism similar to PTPs, 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. detectable with CEL-1(222-623) but not CEL-1(222-585) (Fig. These results again show that the residues 586-623 are important for CEL-1 GTase activity.


Introduction
Most eukaryotic and viral mRNAs are modified at their 5' end by a "cap" structure which 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, m 585) (3, listed as pBS-CEL-1 therein) was used as template. Mutations were verified by dideoxy-DNA sequencing.
The product was digested with Nco I and Sac I and subcloned into pAD5.
PCR-mediated site-directed mutagenesis was used to change Asp112 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 -Mce full-length protein and the RTPase domains of Mce and CEL-1 were expressed using a T7 promoter/polymerase system (19). E. coli strain BL21(DE3) was transformed with the appropriate expression plasmids and cultured in 500ml media at 37 o C to an OD 600 = 0.5. The proteins were induced as described (18).   (8). However, we observed that residual amounts of TEA-HCO 3 2contaminating the purified substrate inhibit RTPase activity, so the protocol was modified as indicated.

RNAi analysis -
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)(30)(31)(32)(33). All of these proteins contain motifs found in the GTase proteins/domains of yeast and virus (2, 28, 34-36; Fig. 2A) as well as an N-terminal domain related to the PTPs.
The protein encoded by 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 8; designated residues 1-236 in those papers). However, there is an in-frame initiation codon 36base upstream of the one previously believed to be the translation start site (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 weight 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 (
Although full-length CEL-1 can simultaneously replace yeast Ceg1 and Cet1, we tested whether over-expression of CEL-1 derivatives could rescue cet1¨VWUDLQ<6%  (47). Outside the active site motif, a conserved aspartic acid residue serves to stabilize the leaving group (48)(49)(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's tyrosine residue.
In order 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. Arginine142 in the consensus motif was mutated. Also, Aspartate 76, Glutamate 111, and Aspartate 112 were also mutated because they were candidates for the proton-donating acidic residue. Arg142 and Asp76 are conserved in all of the PTP-like RTPases, while Glu111 and Asp112 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 [γ-32 P]GTPterminated 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, of that the wild-type protein.
We also tested if these mutants can support viability of a ceg1 ¨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.
Residues Glu111 and Asp112 are not highly conserved and do not seem to be vital in vivo (Fig. 6C). In contrast, Asp76 is conserved and a D76N mutant can not 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 (Asp66 of Mce and Asp60 of BVP) only slightly diminished the activity (51)(52)(53).
Therefore, in contrast to the PTPs, general acid catalysis may not be essential for the mechanism of the RNA phosphatases.
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 Next, we tested the effect of RNA chain length (Fig. 8). CEL-1(13-221) hydrolyzes the beta-gamma 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 k cat /K m values with ATP, dinucleotide, trinucleotide, and tetranucleotide were calculated to be 5.5 cel-1(RNAi) embryos arrested development after forming approximately 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 to 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- 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 α-PSer5 and α-PSer2 for clarity ( Fig. 9C and D).
In the early C. elegans embryo, the appearance of both α-PSer5 and α-PSer2 staining depends upon transcription. Staining with α-PSer5 and α-PSer2 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).
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 α-PSer2 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. 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 (3,8,41,64,51,55,65).   shows that Asp66 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 gamma-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)(69)(70)(71)(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.
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 speculated 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. 4A and B). As 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 (76 and this study). In contrast, a low copy plasmid of the full-length enzyme was sufficient for rescuing a cet1∆ 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.
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 while 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-    were stained with DAPI, a CBP-1 antibody for a staining control (82), and an antibody to CTD phosphoserine 2 (H5) (83,84). An expanded α-PSer2 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 α-PSer2 in the absence of the Pol II epitope (23,63), are marked with asterisks. Representative embryos of comparable stages are shown.
by guest on March 24, 2020