The 1.85 Å Structure of Vaccinia Protein VP39: A Bifunctional Enzyme That Participates in the Modification of Both mRNA Ends

Abstract

VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap-specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 A crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis data suggest two possible RNA-binding sites with novel underlying architecture, one of which forms a cleft spanning the region adjacent to the methyltransferase active site. This report provides a prototypic structure for an RNA methyltransferase, a protein that interacts with the mRNA 5' cap, and an intact poxvirus protein.

Cloning and Characterization of 4EHP, a Novel Mammalian eIF4E-related Cap-binding Protein

“…Thus, we conclude that the mechanism of cap recognition is similar for eIF4E and 4EHP. The structure of eIF4E and that predicted for 4EHP differs markedly from that of the vaccinia protein VP39, which also binds specifically to the cap structure (52,53). However, VP39 also sandwiches the cap structure between two aromatic side chains (phenylalanine and tyrosine) (53).…”

The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs

“…In vitro genetic restoration of tRNA ser Gm18 biosynthesis+ A: Schematic representation of tRNA Ser secondary structure with indication of its natural nucleotide modifications+ B: In vitro synthesized tRNA Ser , labeled by incorporation of a-32 P-UTP, was incubated with S100 extract prepared from wild-type strain (left), TRP1::TRM3 mutant (center), or TRP1::TRM3 transformed with pFL38-PLJ1 expressing Trm3p (right)+ After incubation labeled tRNA was completely digested by either RNAse T2 (top) or nuclease P1 (bottom) and a two-dimensional TLC carried out in system B and A, respectively+ The location of the unlabeled 59 monophosphate nucleotide markers is indicated (dotted spots)+ The extent of modification at the particular positions that can be monitored in each assay is indicated below the chromatograms, expressed as moles of modified nucleosides per mole of tRNA (values were derived from a quantification of spot intensities by a Fuji Bas-1000 Imager)+ ported as yet, will require purification of the protein+ Required both in vivo and in vitro for a single tRNA ribose methylation, Gm18, TRM3 corresponds to the the yeast homolog of E. coli spoU, required for the formation of this modification in all Gm18-containing E. coli tRNAs (Persson et al+, 1997)+ The best studied RNA 29-O-ribose methyltransferase is a viral protein, VP39, the crystal structure of which has been recently resolved (Hodel et al+, 1996)+ VP39 is a bifunctional protein, which can also serve as a poly(A) polymerase processivity factor in addition to its role as 29-Omethylase specific for the penultimate nucleotide of the mRNA 59 cap structure (Shi et al+, 1996)+ In the context of an organism with a rapid generation time, VP39, instead of acquiring an independent RNA binding domain, seems to have customized a methyltransferase catalytic domain for that purpose (Hodel et al+, 1996), and its structure may substantially depart from a consensus for RNA ribose methylases of free living organisms+ In line with this notion, the VP39 sequence was not retrieved in our genomic search+ Likewise, the fact that YDL112w is the only ORF detected in the entire S. cerevisiae genome must reflect an extensive sequence divergence of the other, still unknown yeast 29-Oribose methyltransferases, thus illustrating the limitations of a homology-based approach+ Sequence searches in less stringent conditions might, however, point to additional yeast candidates, although such anal-FIGURE 7. Mutations in tRNA ser affecting Gm18 biosynthesis in vitro+ Mutations preventing the formation of tertiary interactions (thin lines over the cloverleaf structure) were introduced in the T7 tRNA Ser transcript and the modification levels (indicated below and expressed as moles of modified nucleosides per mole of tRNA) were monitored as in Figure 6+ Nucleotides that have been mutated are indicated in circles+ The transcript, labeled by incorporation of a-32 P-UTP, was incubated in the presence of wild-type strain extract and analyzed by 2D-TLC with system B after digestion with RNase T2+ yses might be considerably hampered by high numbers of false positives+…”

“…The three other eukaryotic positive ORFs with very long N-terminal extensions, in human, C. elegans, and A. thaliana, do not necessarily represent orthologs of Trm3p+ Intriguingly, the human ORF encodes a nuclear protein that binds with both high affinity and marked specificity to TAR RNA of HIV, hence its name of TRP-185 for 185-kDa TAR RNA protein (Sheline et al+, 1991;Wu et al+, 1991)+ Its binding is strongly dependent on the TAR RNA loop sequences and is stimulated by a set of cellular factors that also stimulate the binding of RNA polymerase II to HIV TAR RNA+ Binding of TRP-185 and RNA polymerase II to TAR RNA is mutually exclusive and TRP-185 stimulates gene expression from the HIV LTR in vitro (Wu-Baer et al+, 1995a, 1995b, 1996+ Interestingly, defective assembly of large ribosomal subunits in Pet56 mutants seems caused by inactivation of the Pet56 protein rather than by the lack of 21S mitochondrial rRNA G2270 methylation normally catalyzed by this protein, suggesting an additional role for the Pet56 methylase, possibly as a rRNA chaperone (Mason et al+, 1996)+ Likewise, although the presence of Dim1p, which catalyzes formation of the m 2 6 m 2 6 A doublet at the 39 end of 18S rRNA, is a critical factor for pre-rRNA processing, the absence of rRNA base-dimethylation itself does not result in any clear rRNA processing defect (Lafontaine et al+, 1995)+ Moreover, the gene for tRNA m 5 U54-methyltransferase is essential for viability in E. coli, although the known catalytic activity of the methylase is not (Persson et al+, 1992), and the vaccinia virus VP39 29-O-ribose methylase is a bifunctional protein (Shi et al+, 1996), like several other polypeptides (Smith, 1994;Henikoff, 1987)+ In this context, the presence of a long N-terminal extension in the yeast tRNA (Gm18) methylase is consistent with Trm3p having a more complex role than that of a mere RNA modifying enzyme+…”

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

“…The presence of the reducing agent dithiothreitol (DTT; 5 mM) stabilized NS5MTase DV during long time-course experiments (data not shown). In all subsequent tests of NS5MTase DV on 7Me GpppAC n and GpppAC n , we used 40 mM Tris/HCl (pH 7.5) and 5 mM DTT in the absence of MgCl 2 .Interestingly, a similar bell-shaped pH curve with an optimum at 7.5 and independence from divalent cations was found for vaccinia virus mRNA cap 29O-MTase VP39 (Barbosa & Moss, 1978) containing the same catalytic tetrad, K-D-K-E (Hodel et al, 1996(Hodel et al, , 1998. Nevertheless, the optimum conditions for NS5MTase DV described here are in contrast to a reported pH optimum of 29O-MTase activity of NS5MTase DV on another type of short RNA substrate, GpppAGAACCUG, reported recently (Kroschewski et al, 2008;Lim et al, 2008).…”