Evolution of Methyltransferase-Like (METTL) Proteins in Metazoa: A Complex Gene Family Involved in Epitranscriptomic Regulation and Other Epigenetic Processes

Abstract

The methyltransferase-like (METTL) proteins constitute a family of seven-beta-strand methyltransferases with S-adenosyl methionine-binding domains that modify DNA, RNA, and proteins. Methylation by METTL proteins contributes to the epigenetic, and in the case of RNA modifications, epitranscriptomic regulation of a variety of biological processes. Despite their functional importance, most investigations of the substrates and functions of METTLs within metazoans have been restricted to model vertebrate taxa. In the present work, we explore the evolutionary mechanisms driving the diversification and functional differentiation of 33 individual METTL proteins across Metazoa. Our results show that METTLs are nearly ubiquitous across the animal kingdom, with most having arisen early in metazoan evolution (i.e., occur in basal metazoan phyla). Individual METTL lineages each originated from single independent ancestors, constituting monophyletic clades, which suggests that each METTL was subject to strong selective constraints driving its structural and/or functional specialization. Interestingly, a similar process did not extend to the differentiation of nucleoside-modifying and protein-modifying METTLs (i.e., each METTL type did not form a unique monophyletic clade). The members of these two types of METTLs also exhibited differences in their rates of evolution. Overall, we provide evidence that the long-term evolution of METTL family members was driven by strong purifying selection, which in combination with adaptive selection episodes, led to the functional specialization of individual METTL lineages. This work contributes useful information regarding the evolution of a gene family that fulfills a variety of epigenetic functions, and can have profound influences on molecular processes and phenotypic traits.

Keywords: METTL; epigenetics; metazoan; methyltransferase; phylogenetics; selection.

Fig. 1.

METTLs in major metazoan phyla according to NCBI search results. The metazoan cladogram is modified from the Tree of Life web project (http://tolweb.org/tree/phylogeny.html). The matrix displays the detection (gray), probable detection but with uncertain identification (striped-gray), or failure of detection (white) by the search for each METTL gene within each phylum. The number of METTL genes out of 33 total is noted in parenthesis with asterisks indicating phyla in which probable METTL sequences were either located but could not be definitively identified as specific METTL genes, or were located outside of our search in the NCBI GenBank database (e.g., Ensembl Metazoa). METTLs are listed in order of decreasing number of representatives in each phylum (left to right).

Fig. 2.

Maximum likelihood tree describing the evolutionary relationships across metazoan METTL proteins. Bootstrap values (1,000 replicates) are displayed at each internal node. Branch labels displayed in blue indicate METTLs that have been shown to target DNA or RNA nucleosides, labels in red are METTLs shown to target protein residues, and labels in purple represent METTLs whose function and targets are unknown. The METTL proteins comprise nine different taxa representing eight different phyla: A sponge (Amphimedon queenslandica, Porifera), a stony coral (Acropora millepora, Cnidaria), a sea hare (Aplysia californica, Mollusca), a priapulid (Priapulus caudatus, Priapulida), a shrimp (Litopenaeus vannamei, Arthropoda), a sea star (Acanthaster planci, Echinodermata), an acorn worm (Saccoglossus kowalevskii, Hemichordata), a lancelet (Branchiostoma belcheri, Chordata), and human (Homo sapiens, Chordata). Subtrees with the same METTL gene from multiple taxa have been collapsed for figure readability.

Fig. 3.

Maximum likelihood phylogeny reconstructed based on the CDs of each METTL protein across Metazoa. Bootstrap values (1,000 replicates) are displayed at each node. Branch label colors are identical to figure 2.

Fig. 4.

Estimated rates of evolution of METTL proteins. Rates were only estimated for METTLs in which amino acid differences per site could be calculated by comparing the protein between three or more of the preselected metazoan phyla.

Fig. 5.

Episodes of diversifying selection shaping the evolution of METTL proteins in vertebrates. For figure readability, “METTL” has been removed from the names of each tip label (e.g., “METTL1” is simply displayed as “1”). (A) The strength of selection at significant branches is represented in red (ω > 5), gray (ω = 1), and blue (ω = 0), with the proportion of sites within each class represented by the color width. Thicker branches have been classified as undergoing episodic diversifying selection at corrected P value < 0.001 (thicker branches) and P value < 0.01 (thinner branches). (B) The physical positions of adaptive selection episodes involved in the diversification of METTL genes. Numbers of synonymous (blue bars) and nonsynonymous (red bars) substitutions at codon positions that are subject to significant episodes of diversifying selection in vertebrates (P value < 0.05). (C) Phylogenetic location of the mutations involved in such episodes. Branches in red account for higher numbers of nonsynonymous mutations, branches in blue indicate higher numbers of synonymous mutations, and branches in green represent cases with the same numbers of nonsynonymous and synonymous mutations. Light blue, light red, and light purple backgrounds behind each branch indicate METTLs that modify DNA/RNA, proteins, or unknown targets, respectively. Subtrees that include several closely related METTLs (i.e., METTL11A and METTL11B; METTL21A, METTL21B, METTL21C, METTL21D, and METTL21E; METTL25 and METTL25B) were collapsed into a single branch for figure readability.

Fig. 6.

Episodes of adaptive selection identified upon separately analyzing vertebrate DNA/RNA-modifying METTLs and vertebrate protein-modifying METTLs. (A) The physical positions of adaptive selection episodes involved in the diversification of METTLs that modify DNA/RNA nucleosides (top) and METTLs that modify protein residues (bottom). Numbers of synonymous (blue bars) and nonsynonymous (red bars) substitutions at codon positions that are subject to significant episodes of diversifying selection in vertebrates (P value < 0.05). (B) Phylogenetic location of the mutations involved in such episodes for DNA/RNA-modifying METTLs, indicated by a light blue background, and (C) protein-modifying METTLs, indicated by a light red background. For figure readability, “METTL” has been removed from the names of each tip label (e.g., “METTL1” is simply displayed as “1”). Branches in red account for higher numbers of nonsynonymous mutations, branches in blue indicate higher numbers of synonymous mutations, and branches in green represent cases with the same numbers of nonsynonymous and synonymous mutations.