Wyosine derivatives are tricyclic ribonucleosides present exclusively at position 37 of tRNA specific for phenylalanine in most Eukarya (cytoplasmic only) and Archaea, but not Bacteria. They occur by posttranscriptional modification of encoded guanosine in the tRNA precursor. Depending on the organism examined, eight wyosine derivatives have been identified in naturally occurring tRNA^Phe, eleven if one also includes biosynthetic intermediates. The large diversity of wyosine derivatives attests to the existence of complex biosynthetic routes that differ from one organism to another. In this review, we describe the chemical structures of these hypermodified guanosine derivatives and the biosynthetic pathway of the wyosine derivative found in Saccharomyces cerevisiae. Not surprisingly given their location at position 37, 3′adjacent to the GAA anticodon, wyosine derivatives have been demonstrated to play an essential role in translation, particularly in the regulation of frameshifting.

Introduction

All transfer RNA genes sequenced so far harbor a purine (A or G) at position 37, adjacent to the 3′side of the anticodon.^1,2 After transcription this purine is often enzymatically modified into more complex derivatives, the nature of which depends on the sequence of the anticodon and the organism,^3,4 (recently reviewed in refs 5 and 6) recently reviewed in refs. 5 and 6. In the case of tRNA^Phe (anticodon GAA, 31 sequences deposited in the tRNA databank—http://trnadb.bioinf.uni-leipzig.de/), two sets of unrelated compounds are found: i) isopentenyladenylate derivatives, such as isopentenyladenosine (i⁶A) in the cytoplasmic tRNA^Phe of some eukaryotes and methylthiolated i⁶A and/or hydroxylated derivatives (io⁶Ams²i⁶Ams²io⁶A) in tRNA^Phe of Bacteria and eukaryotic organelles; ii) a simple N¹-methylguanine (m¹G) in tRNA^Phe of some Bacteria, Archaea and Eukaryotes and more complex derivatives of the hypermodified nucleoside wyosine (in fact of demethylwyosine, the minimalist derivative in this series, see below) in the cytoplasmic tRNA^Phe of other Eukarya⁷ and Archaea (Fig. 1A and below). Recently, Bujnicki and coworkers have analyzed in great detail the occurrence and biosynthetic pathway of the isopentenylated adenosine derivatives in 63 organisms.⁸ Here, we focus on the structure and the biosynthetic pathway of the other family of purine-37 derivatives, wyosines.

Figure 1

Wyosine derivatives identified in naturally occurring tRNA^Phe of Eukarya (from C to G) and Archaea (from G to J). Names and symbols are the conventional ones adopted from scientific literature. Numbering of atoms depends on whether it is used for the (more...)

Discovery of the So-Called ‘Y’ Base

In the flurry of excitement over the purification and sequencing of tRNAs, scientists in the nineteen sixties observed some peculiarities in one fraction of nucleic acids containing a tRNA specific for phenylalanine and isolated from baker's yeast or animal liver. This eukaryotic tRNA^Phe was found to contain a highly fluorescent nucleoside.^9-11 The presence of this peculiar base accounts for the remarkable hydrophobic behavior of tRNA^Phe during column chromatography¹² and has been shown to be essential for proper codon-anticodon interaction.^11,13 Also, due to the lability of the glycosidic bond this compound was easily excised from tRNA by incubation under mildly acidic conditions (pH 2-4, 37°C for a few hours) without breaking the tRNA phosphodiester backbone or affecting any other bases of the tRNA^Phe.¹⁴ The structure of the acid liberated ‘Y’ base of baker's yeast was subsequently determined to be a tricyclic purine (imidazopurine) now designated wybutine.^14,15 Since the acid treatment might have altered the chemistry of the isolated Y base, its structure was independently confirmed by total synthesis.^16,17 The position of the glycosidic linkage was established by chemical analysis of the nucleoside obtained after mild enzymatic hydrolysis of purified S. cerevisiae tRNA^{Phe 18} and much later verified by chemical synthesis.¹⁹ Wybutosine (yW, Fig. 1C and Table 1), the nucleoside of wybutine, has a UV light absorption maxima at 234, 263 and 310 nm and a fluorescence emission maximum at 443 nm with excitation maxima at 239 and 318 nm at pH 7.5.²⁰ In view of the steric hindrance between the N4-methyl group and the ribose moiety, it is likely that yW exists in tRNA exclusively in the anti conformation (not in syn conformation as it is drawn in Fig. 1C), a conformation that has been confirmed in solution for the nucleoside by NMR²¹ and in the solid state by crystallography.²² Wyosine derivatives are highly photoreactive, ²³ a characteristic that has to be taken into account when testing the function of tRNA^Phe. In this chapter, we use the numbering of the atoms as defined by Blobstein and coworkers²⁴ for the tricyclic compounds when the imidazole is fused to the purine. This numbering differs from the IUPAC convention used for purines (compare the numberings in the Figs. 1A and 1C).

Table 1

Wybutine and wyosine derivatives found at position 37, 3′adjacent to anticodon GAA of tRNA^Phe in Eukarya (E) and Archaea (A).

Other Members of the Wyosine Families in Eukaryota

Experiments aimed at identifying the structures of the modified purine-37 in tRNA^Phe were carried out on a large panel of eukaryotes, including the yeast Torulopsis utilis, animal liver (bovine, rat, chicken, rabbit), several plants (wheat germ, yellow lupine and maize seeds), insects (Drosophila melanogaster and Bombix mori), firmicute Mycoplasma kid and the aquatic fungus Geotrichum candidum. While a fluorescent tricyclic imidazopurine was detected in most cases, it was clear that the precise chemical structure was dependent on the organism and the method of isolation.

Interestingly, the structure of wyosine derivatives isolated from plants or animal liver were first reported to be β-hydroperoxywybutosine (o2yW, Fig. 1D and Table 1), the only peroxy group within the lateral amino acid chain of wybutine.^24-29 The same structure was later reported in tRNA^Phe isolated from the aquatic fungus Geotrichum candidum.³⁰ The presence of a hydroperoxide in tRNA^Phe from L. luteus and wheat germ was supported by a positive Fe(SCN)₂ coloring test.^28,29 However, based on a negative Fe(SCN)₂ coloring test and mass spectrometry data, Itaya and coworkers suggested that a hydroxyl group (OHyW, Fig. 1E and Table 1) instead of a hydroperoxide group was present in wybutosine of rat liver tRNA^{Phe 31}, and later synthesized OHyW.^32,33 However, because hydroperoxywybutosine (o2yW) can be formed during storage or manipulation of hydroxywybutosine (OHyW)³³ and decomposition of o2yW leads to the formation of hydroxywybutosine (OHyW) and wybutosine (yW),²⁹ the ultimate resolution of the relevant structure(s) may require the identification of the corresponding metabolic enzyme(s) forming one or another of these two experimentally identified wyosine derivatives.

A simpler wyosine derivative, designated wyosine (imG), has been isolated and characterized from tRNA^Phe of the yeast Torulopsis utilis (alternative names Candida utilis, Torula utilis, Pichia jadinii).^34,35 Its structure (Fig. 1G and Table 1) has been unambiguously verified by several methods, including comparison with the chemically synthesized nucleoside.¹⁹ Like the wyosine derivatives described above, imG is characterized by the susceptibility of the glycosidic bond to acid-catalyzed hydrolysis³⁴ and remarkable fluorescence when illuminated with a UV-lamp.³⁵

Finally, intermediates of wybutosine biosynthesis have been isolated from a variety of cell types. For example, when Vero cells (a cell line derived from African green monkey kidney) are grown in medium deprived of methionine (Met starvation), the yW (normally present in an important fraction of the cellular tRNA^Phe) is absent; addition of methionine to the growth medium leads to reappearance of tRNA^Phe fully modified with wybutosine.³⁶ The tRNA^Phe isolated from various tumor cells (e.g., Ehrlich ascites, neuroblastoma and Novikoff hepatoma) has been shown to be hypo-modified at the lateral chain (OHyW*, Fig. 1F and Table 1) and contain a fraction of tRNA^Phe with 1-methylguanosine at position 37 (m¹G, Fig. 1B).^37-39 The presence of m¹G in tRNA^Phe has also been observed in a number of other cell types and organisms, including rat liver hepatomas,⁴⁰ the previtellogenic oocyte of amphibian Tinca tinca,⁴¹ the posterior silkgland of Bombyx mori,⁴² Mycoplasma kid⁴³ and from Drosophila melanogaster.^44,45

Wyosine Derivatives also Exist in Archaea

To date, the only tRNA^Phe that has been sequenced from an archaeal organism originates from the euryarchaeon H. volcanii: it harbors an m¹G37.⁴⁶ However, wyosine derivatives have been identified in the tRNA^Phe of other Archaea by analysis of tRNA enzymatic digests with combined liquid chromatography-mass spectrometry (LC-MS). The first wyosine derivative was found in digests of the hyperthermophilic crenoarchaeota Sulfolobus solfataricus, Thermoproteus neutrophilus and Pyrodictium occultum. Based on the comparison of the UV absorption spectrum, fluorescence properties and mass spectrometry with those of eukaryotic wyosine derivatives and of synthetic bases, a new wyosine derivative was identified⁴⁷ as 7-methylwyosine (mimG, Fig. 1H). However, further analysis of eleven additional thermophilic Archaea, including phylogenetically diverse representatives of thermophilic methanogenes and sulfur metabolizing hyperthermophiles of the euryarchaeota group (e.g., Methanobacterium thermoautotrophicum, Thermoplasma acidophylum and Archaeoglobus fulgidus), revealed that only a few Archaea (mainly the crenoarchaeota) contain mimG in their tRNAs,⁴⁸ albeit demethylwyosine, which lacks the methyl group at N4, can also be detected. In enzymatic tRNA extracts of Sulfolobus solfataricus P2, Methanococcus thermolithotrophicum and Stetteria hydrogenophila, two new compounds have been identified: one corresponds to an isomer of wyosine (imG) and is designated isowyosine (imG2, Fig. 1I and Table 1), while the other one is a minimalist form of imG/imG2 with a molecular mass of 321, designated imG-14 because its molecular mass is 14 Da less than that of wyosine imG (Mr = 335, Fig. 1J and Table 1).⁴⁹ Pyrolobus fumarii, a submarine crenarchaeote which grows optimally at 106°C, has been shown to contain several wyosine derivatives, including mimG, imG2, imG-14 and imG (the same as in T. utilis—Fig. 1G).⁵⁰ The imG-14 (not mimG and imG/imG2) has also been detected in the psychrotolerant archaeon Methanococoides burtonii, which grows at 4-23°C,⁵¹ demonstrating that imG-14 derivatives in archaea are not exclusively synthesized in thermophilic or hyperthermophilic organisms. A compound of yet unknown structure with Mr = 422, exhibiting UV absorption spectrum characteristic of wyosine derivatives (designated N422), has been found in M. maripaludis and M. vannielii⁵² but not in the other archaea analyzed so far.

Thus, depending on the archaeon, at least four imG-14 derivatives can be identified in hydrolysates of bulk tRNA (imG, imG2, mimG (see Fig. 1, G-J and possibly N422). Their structures appear to be unique to Archaea because of the presence of a simple methyl group (or none) at C7 of the imidazopurine (Figs.1H and 1I) instead of α-amino-α-carboxypropyl side chain as in eukaryal wybutosines (Fig. 2, C-F). Unlike all eukaryal wyosine derivatives identified so far, archaeal derivatives do not always harbor a methyl group at N4.

Figure 2

Wybutosine biosynthesis pathway. The various steps leading to the formation of wybutosine (yW) in the yeast S. cerevisiae (taken from ref. ), yeast Torula utilis and plants/ higher eukaryotes are shown. For the enzymatic steps where the cofactors involved (more...)

Biosynthesis of Wyosine Derivatives in Eukarya

The Biosynthetic Route of Wybutosine Biosynthesis in S. cerevisiae

Integration of all the above information allows the following scheme to be proposed for the multistep enzymatic formation of wybutosine derivatives (Fig. 2). The complete biosynthetic route encompasses 7 steps (most taken from ref. 67): (1) Formation of m¹G37 catalyzed by tRNA:m¹G methyltransferase (Trm5p). The crystal structure of only the archaeal homolog (Methanocaldococcus jannaschii⁶²) is known and raises interesting questions about the mechanism of action of this AdoMet-dependent methyltransferase (for the human homologue Trm5p, see also;⁶⁸ (2) Addition of a C₂-unit followed by cyclization of the 3rd ring catalyzed by Tyw1p. This enzyme contains a flavodoxin domain in its N-terminus and a radical-AdoMet domain including one (or possibly two) (4Fe-4S) cluster(s) in its C-terminus. The FMN cofactor in the flavodoxin domain probably serves as an electron storage unit. Mutations in one of the (4Fe-4S) cluster motifs abolished the yW biosynthesis in vivo, demonstrating that it is essential for catalytic activity.⁶⁷ Crystal structures of the archaeal Tyw1p homologues lacking the N-terminal flavodoxin domain have recently been solved from M. jannaschii and Pyrococcus horikoshii.^69,70 Their overall structures are similar to other radical-AdoMet enzymes^71,72 and are consistent with the predicted enzymatic activity for imidazo ring formation (for details see chapter by Atta et al in this volume). The identity of the two-carbon donor in this reaction remains to be identified; (3) Transfer of the α-amino-α-carboxypropyl group (acp) from the methionine moiety of AdoMet to imG-14 at C7 atom catalyzed by Tyw2p. This is consistent with the earlier observation that the acp moiety of yW originates from methionine;⁵⁵ (4) AdoMet-dependent methylation of the yW-86 (7-aminocarboxypropyl-demethylwyosine) to give yW-72 (7- aminocarboxypropylwyosine) by Tyw3p. In S. cerevisiae it appears that the Tyw2p activity occurs before Tyw3p and not vice versus, while in T. utilis imG is the apparent final modified nucleoside, demonstrating that in some organisms Tyw3p can catalyze the AdoMet-dependent conversion of imG-14 to imG (Fig. 2, dashed arrow); (5,6) The AdoMet-dependent conversion of yW-72 (aminocarboxypropylwyosine) to yW-58 (aminocarboxypropylwyosine methyl ester) and further to yW (methoxycarbonyl-aminocarboxypropylwyosine methyl ester), catalyzed by Tyw4p. Only scanty amount of yW-58 intermediate is formed in vitro. Still, it demonstrates that recombinant TYW4p has an apparent Ado-Met-dependent methylation activity. The fact that yW is a major product of TYW4p-catalyzed reaction together with the absence of detectable yW-14 or other possible intermediates indicate that multistep enzymatic formation of yW from yW-72 is most probably triggered by Ado-Met-dependent methylation. Since small quantity of other protein involved in methoxycarbonylation of yW-58 might copurify with the his-tagged TYW4p protein by a Ni-chelating column, TYW4p was further purified by anion exchange chromatography. Such purified protein directly converts yW-72 to yW with production of only low amount of yW-58 intermediate, suggesting that TYW4p is a bifunctional enzyme catalyzing both methylation of α-carboxyl group and methylcarbonylation of the α-amine group in the lateral chain of wyosine. (7) Hydro(pero)xylation of wybutosines. The enzymes catalyzing such reactions in plants and higher eukaryotes are unknown. Altogether, at least five molecules of AdoMet are consumed in the biosynthesis, making wybutosine perhaps the most costly modified nucleotide formed in a single cellular tRNA.⁷³ To conclude this part of the chapter, Figure 3 summarizes the present knowledge about the origin of various ‘building blocks’ of wyosine derivatives in Eukarya and Archaea.

Figure 3

Origins of ‘building blocks’ in wybutosine. This figure complements Figure 2 concerning the biosynthetic pathway. Various ‘building blocks’ of wyosine derivatives in Eukarya and Archaea are presented.

Role of Wyosine Derivatives During Translation Process

The high-energetic cost and the requirement of many specific enzymes for its biogenesis beg the question of the cellular function of wybutosine in tRNA^Phe. Its location exclusively at position 37, 3′-adjacent to the GAA anticodon, suggests a function in the decoding process on the ribosome. However, since there is significant structural diversity in the wyosine deriviatives found in Eukarya and Archaea (and Bacteria completely lack wyosine derivatives), how these nucleosides might participate in decoding is not immediately obvious. It appears from many studies that, in general, the modification status of tRNA^Phe, especially at purine-37, plays a role in modulating the stability of the codon-anticodon interaction by dangling end-type base stacking (stabilization function⁷⁴). Also, because most modified purines at position 37 cannot base pair in a Watson-Crick mode, their presence 3′-adjacent to the anticodon restricts the tRNA to base pair with the in-frame codon-anticodon triplet, thus limiting (but not completely avoiding, see below) the risk of frameshifting during translation (‘antislip’ function, reviewed in refs. 6,75,76). In the special case of tRNA^Phe (anticodon GAA), which has to read ‘potentially weakly binding’ UUU/C codons, sometimes in a run of ‘slippy U-rich’ codon contexts, there may be a particular need for the ‘stabilization and antislip’ functions of highly hydrophobic bases like the wyosine derivatives. Notably, the level of modification at purine-37 and especially of the wybutosine derivatives in eukaryal tRNA^Phe, depends on cell growth or stress conditions and the availability of the cofactor(s) needed for their enzymatic formation (see references above). Thus, the presence or absence of wybutosine derivatives in tRNA^Phe may affect and possibly regulate, the expression level of certain proteins. For example, in several retroviruses the production of gag-pol or pro-pol fusion proteins requires -1 frameshifting at the so-called ‘programmed frameshifting’ site U-UUU-UUA (the slippery sequence read by tRNA^Phe is underlined).⁷⁷ Interestingly, the tRNA^Phe isolated from HIV infected H9 cells lacks the wyosine derivatives.⁷⁸ Also, using a rabbit reticulocyte in vitro translation system and the ‘shifty’ A-AAU-UUU sequence, Hatfield and collaborators demonstrated that the rabbit reticulocyte tRNA^Phe bearing a m¹G37 instead of the yW37 stimulates -1 frameshifting fourfold.^79,80 However, when tRNA^Phe from the S. cerevisiae trm5 mutant, which contains G37, was tested in vivo with another slippery sequence U-UUU-UUA, no significant difference in the level of frameshifting was observed when compared with the wild type tRNA^Phe containing yW37.⁸¹ These contradictory observations indicate that either the consequences of a lack of yW37 depend on the position of the U-UUU sequence in the heptameric shifty sequence, or the two biological systems (in vitro or in vivo) are not comparable. Recently, Schimmel and coworkers demonstrated a clear correlation between the degree of modification of the G37 in yeast tRNA^Phe and the efficiency of -1 frameshifting.⁸² Using an in vivo system based on mutants of S. cerevisiae ΔTYW1 and ΔTYW2 that accumulate tRNA^Phe bearing m¹G or imG-14, respectively (see Fig. 2), they probed all six naturally occurring shifty sequences in SCV-LA virus: (G-G(G,A,U)U-UU(U,C)), followed by a characteristic pseudo-knot that was shown to favor the frameshift event. The results were very compelling; tRNA^Phe bearing m¹G37 was more prone to frameshifting (up to 35% in some cases) than the one bearing imG-14 at position 37 (about 25%), which itself was more prone to frameshift than the wild type tRNA^Phe containing yW37 (around 15%). It now remains to explain these data at the molecular level and importantly, to understand why some types of cells do not contain wyosine derivatives and have m¹G37 instead.

Conclusion and Future Prospects

Wyosine and related derivatives are found exclusively at position 37, 3′-adjacent to anticodon GAA in tRNA^Phe of many Eukarya and Archaea. They are absent in tRNA^Phe of Bacteria, which instead harbor m¹G37. In Figure 4, arrows indicate the direction of chemical complexity of different derivatives found in the three phylogenetic domains, from simple m¹G to the more elaborate imG-14, 7-methylwyosine (mimG) in Archaea and wybutosine derivatives (yW and o2yW/OHyW) in Eukarya. Biosynthesis of these guanosine derivatives, especially in higher eukaryotes and plants, involves many enzymatic steps and is energetically costly in terms of AdoMet consumption. Its function is clearly to facilitate and regulate the production of proteins at the translation level (stabilization of codon-anticodon interaction and avoidance of slippage out of reading frame). However, there is also evidence that wyosine biosynthetic enzymes might be associated with other functions. For example, overexpression of the TRM12 gene, which encodes the human homologue of Tyw2p (step 3 in Fig. 2), has been observed in human breast cancer cells.⁸³ Since it has been previously observed that in some tumors a hypomodified tRNA^Phe (bearing m¹G37) is utilized in translation instead of the fully modified isoacceptor,⁸⁴ overproduction of this tRNA modification enzyme may trap a fraction of the imG-14 harbouring tRNA^Phe, leaving free the m¹G37-bearing variant. Thus, exploring the origin of the overexpression of TRM12 in cancer cells may reveal novel pathways of tumorogenesis.

Figure 4

Distribution of modified purines found in tRNA^phe in organisms of the three domains of life. Symbols are explained in the text. Arrows indicate the direction of chemical complexity. The first basic transition, common to both eukaryal and archaeal organisms, (more...)

While considerable progress has been made in recent years elucidating the wyosine biosynthetic pathway, much remains to be done. For example, the two-carbon donor for the radical-AdoMet enzyme Tyw1p (step 2 in Fig. 2) has yet to be identified, as well as the donor of methylcarbonyl (step 6 in Fig. 2) and the enzyme(s) involved in the hydro(peroxy)lation of wybutosine (step 7 in Fig. 2) in plants and higher eukaryotes are still unknown. Moreover, the potential exists for the organization of wyosine biosynthetic enzymes into a multiprotein complex (metabolon).⁶⁷ At least in plants, genes coding for homologues of Tyw2p, Tyw3p and the C-terminal domain of Tyw4p are fused and code for a large ‘TYW3-4C-2’ protein.⁶⁷ The close proximity of enzymes active-sites and substrates within multi-enzyme complexes generally enables the reactions to proceed more efficiently and without accumulation of intermediates. Crystallization of such type of complex should shed light about how the successive reactions are performed in a sequential manner. The three-dimensional structures of several wyosine and wybutosine biosynthetic enzymes have been solved, but so far only for archaeal homologues.^62,69,70

Presently, the set of genes encoding enzymes of the wybutosine biosynthetic pathway is known only in S. cerevisiae. There are several different imG-14 derivatives in eukaryal and archaeal tRNA and searching for homologous genes in fully sequenced genomes (53 archaeal and 21 eukaryal ones are available at http://img.jgi.doe.gov, August 2008) and correlating them with the presence of imG-14 derivatives in each of these organisms may reveal the existence of alternative metabolic routes and provide insight on the emergence and evolution of this huge family of genes.

Acknowledgements

We thank Jef Rozenski (Katholieke Universiteit Leuven, Belgium) for suggestions regarding the nomenclature of wybutosine derivatives and Dirk Iwata-Reuyl (Portland State University, Portland, Oregon, USA) for advices and considerable improvements of the text. Current work on wyosine derivatives in LD laboratory is financed by the Fonds pour la Recherche Fondamentale Collective (FRFC), Fonds Jean Brachet Recherche, Fonds E. Defay and Fonds D. et A. Van Buuren. JA thanks the Commissariat à l′Energie Atomique for financial supports. JU was supported by a postdoctoral fellowship from the FRFC and by a FEBS Distinguished Young Investigator Award. HG (emeritus scientist) thanks Prof. Jean-Pierre Rousset from Université Paris-Sud for providing facilities to continue working in his laboratory.

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