Edited by: Tohru Yoshihisa, University of Hyogo, Japan
Reviewed by: Hiroyuki Hori, Ehime University, Japan; Yohei Kirino, Thomas Jefferson University, United States
†These authors have contributed equally to this work
This article was submitted to RNA, a section of the journal Frontiers in Genetics
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Among tRNA modification enzymes there is a correlation between specificity for multiple tRNA substrates and heteromultimerization. In general, enzymes that modify a conserved residue in different tRNA sequences adopt a heterodimeric structure. Presumably, such changes in the oligomeric state of enzymes, to gain multi-substrate recognition, are driven by the need to accommodate and catalyze a particular reaction in different substrates while maintaining high specificity. This review focuses on two classes of enzymes where the case for multimerization as a way to diversify molecular recognition can be made. We will highlight several new themes with tRNA methyltransferases and will also discuss recent findings with tRNA editing deaminases. These topics will be discussed in the context of several mechanisms by which heterodimerization may have been achieved during evolution and how these mechanisms might impact modifications in different systems.
Critical to substrate binding specificity is the fact that enzymes need to achieve “high” affinity for their targets while ignoring non-targets. What makes this an especially difficult problem in cells is that substrates and non-substrates often look very similar, which tests the limits of enzyme-substrate recognition. This tenet is especially true of RNA binding proteins where high-affinity binding usually involves indirect readout of the phosphate backbone of RNA that must be combined with base- and shape-specific contacts to enable substrate discrimination. Many enzymes follow these rules to achieve effective target specificity, yet some face an additional obstacle, in that they must also maintain the ability to turn over during a catalytic cycle in order to yield a productive reaction. This issue is exacerbated when a single enzyme must target different substrates within a pool of nearly identical ones, as is the case faced by most tRNA modification enzymes.
A growing trend in the modification field is that many of the enzymes which recognize multiple substrates are heteromultimeric (
Certain eukaryotic tRNA methyltransferases strictly function as two-subunit enzymes: where association between a structural and a catalytic subunit is required for tRNA methylation (
Eukaryotic two-subunit dependent methyltransferases. The figure shows examples of methylations catalyzed by heteromultimeric enzymes; a theme with eukaryotic modifications.
The nuclear Trm61/Trm6 complex responsible for
High-affinity binding of Trm61/Trm6 to tRNA substrates, such as tRNAiMet or tRNALys,3UUU, was originally thought to depend primarily on Trm6 as the RNA recognition subunit (
Trm61/Trm6 has been suggested to arise through duplication and divergence from an ancestral TrmI-family enzyme, a family that catalyzes m1A58 formation in bacteria, and/or m1A57 in archaea (
The nuclear complex of Trm8/Trm82, responsible for
Trm8/Trm82 homologs are readily identifiable in yeast, protist, plant, and animal species. No apparent Trm8 or Trm82 homologs are currently found in archaeal genomes, which generally lack m7G46. Bacterial genomes do not contain an obvious Trm82 homolog, however, Trm8 shares sequence similarity with the bacterial TrmB-family (
Comparison of apo-Trm8/Trm82 to its tRNA bound form revealed that, unlike Trm61/Trm6, Trm82 makes no apparent RNA contacts in the context of a Trm8/Trm82 complex (
Trm112 can partner with either Trm9 or Trm11 to form separate SAM-dependent tRNA methyltransferase complexes, which act on separate pools of cytoplasmic tRNA substrates, at different nucleotide sites, and produce distinct chemical products (
Work in
The C-terminus of the multi-domain protein methyltransferase ALKBH8 contains a subdomain with high sequence similarity to Trm9, sufficient in formation of mcm5U34-style modifications at the tRNA wobble position. ALKBH8 contains additional functional domains: an N-terminal RNA recognition motif, an AlkB-related domain, and a zinc finger region upstream to the C-terminal Trm9 orthology region (
The requirement of the
As previously noted Trm11 homologs are absent from bacteria, while Trm9 mcm5U-type modifications are exclusive to eukaryotes, yet Trm112 is broadly conserved across every domain (
The catalytic subunit Trm7, a 2′-
In yeast and other eukaryotes, certain tRNAs contain both Cm32 and Nm34 modifications in tandem. The most broadly conserved tandem methylated substrates are tRNAPhe species. In yeast lack of Trm7-modified tRNAPhe activates the general amino acid control starvation response (
In most organisms, a tRNA sequence will contain well over a dozen post-transcriptional chemical modifications. An evergreen topic of discussion is whether modification enzymes act in a particular order, where modification at one site is informed by the modification status of other positions. In eukaryotes, compartmentalization of modification enzymes obviously results in the sequential order of some chemical transformations. For example, nascent tRNA transcripts are first modified in the nucleus; after export to the cytoplasm, tRNAs can be further modified. Because tRNAs can also be imported into organelles (chloroplast or mitochondria), these may receive other modifications in addition to those already obtained in the nuclear and cytoplasmic compartments. However, the sequential nature of certain tRNA methylation events cannot be explained by compartmentalization alone and may be an inherent property of enzymes that work in complexes or those who have achieved a heteromeric state. Alternatively, selection for increased specificity may be a leading factor in establishing sequentiallity, such may be the case of complex modification pathways such as those for wybutosine and threonylcarbamoyl synthesis.
With few exceptions, tRNA sequences encode for a purine at position 37 that is almost always post-transcriptionally modified. Guanosine at position 37 can be methylated, with more complex conversion to wybutosine (yW) in tRNAPhe (
Chemical deamination of A-to-I in RNA sequences was observed 30 years prior to the discovery of any hydrolytic deaminase responsible for A-to-I activity that could act on polynucleotides (
Adenosine-to-inosine (A-to-I) editing of tRNAs. The figure shows the different types of deamination reactions occurring in tRNAs from all domains of life.
Inosine at the first position of the anticodon (I34) is essential in both eukaryotes and bacteria, but it does not occur in archaea (
A34-to-I34 deamination of eukaryotic tRNAs is catalyzed by the heterodimeric enzyme of (ADAT2/ADAT3 or Tad2/Tad3), which requires association between two paralogous sub-units for activity, while bacteria rely on the homodimeric enzyme ADATa (or TadA) (
I37 and I57 are less widespread within organisms, where I37 is found in tRNAAla of certain eukaryotes (
All polynucleotide deaminases belong to the cytidine deaminase superfamily and require a Zn2+ for activity. However, phylogenetic analysis has revealed that ADAT1 closely aligns with mRNA specific ADARs, while ADAT2/ADAT3, responsible for inosine formation at position 34, strikingly resembles nucleic acid cytidine deaminases such as AID (activation-induced deaminases) or APOBEC (the apolipoprotein B mRNA editing enzyme) (
Certain archaea and eukaryotes contain C-to-U edited tRNAs, but in many instances the enzymes responsible remain to be identified (
Cytidine-to-uridine (C-to-U) editing of tRNAs.
C-to-U editing of tRNAs in eukarya was first reported in protists, plants and marsupial mitochondria (
In the mitochondria of dicotyledon plants, C-to-U editing takes place outside the anticodon region and does not directly influence the decoding capacity of tRNAs (
Position 32 of the anti-codon stem loop is another site where tRNAs from multiple domains contain C-to-U conversions. It was first described in
As discussed in Section “Adenosine to Inosine (A-to-I) Editing” A-to-I editing is found at three tRNA sites: position 34, 37, and 57. Intriguingly, inosines at position 37 and 57 can be methylated to form m1I37 and m1I57, by distinct chemical pathways (
In trypanosomes, down regulation of ADAT2/ADAT3 expression reduces A34-to-I34 editing in tRNAThrAGU at position 34 and C-to-U editing at position 32 (
The interdependence model showing the necessary connection between RNA modification and RNA editing.
ADAT2/ADAT3 can deaminate DNA
Much has been written about the mechanisms that lead to, and determine the fate of, duplicated genes. It is clear that once gene duplication occurs, one copy is free to mutate via genetic drift, to accumulate mutations perhaps by a “neutral evolutionary ratchet” (
In the case of Trm7/Trm732 and Trm7/Trm734, no evidence exists for gene duplication; neither Trm732 nor Trm734 have significant sequence conservation with known methyltransferases. Instead, they share similarity with other protein families, for example Trm734 with WD-domain family proteins. It may be that independent stochastic mutations accumulated in each gene for evolution of a dimerization interface that allowed each protein to interact with the Trm7 partner. In doing so, differential complex formation permitted a novel division of labor; one complex now targets position 32 of tRNAs and the other position 34. This is, of course, assuming that the catalytic subunit Trm7 derives from an ancestral gene that at some point was able to efficiently methylate both positions.
Similar arguments can be made with the tRNA A-to-I deaminase of trypanosomes, TbADAT2/TbADAT3, a heterodimeric enzyme comprised of two subunits encoded by clear paralogs (
Finally let’s consider m3C/m3U editing and methylation at position 32 of several tRNAs in
Neo- or sub-functionalization includes a combination of non-adaptive and, later, adaptive mutations as originally suggested by
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank all members of the Alfonzo laboratory for useful discussions and comments.