Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II

Abstract

Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species. Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists. Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists. Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (RPB1) from the pelobiont Mastigamoeba invertens. Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases. In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs. In contrast with rDNA-based trees, phylogenetic analyses of RPB1 sequences indicate that Mastigamoeba represents an early branch of eukaryotic evolution. Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that Mastigamoeba RPB1 is attracted to the base of the eukaryotic tree artifactually. In addition, the presence of introns and a heptapeptide C-terminal repeat in the Mastigamoeba RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.

Figure 1

Insertion sites relative to conserved domains and terminal sequences for five putative introns found in the Mastigamoeba RPB1 gene. Amino acid residues shown in boldface are conserved and can be aligned among all RPB1 sequences. Dinucleotides coding for putative splice donor and acceptor sites are also in boldface.

Figure 2

Codon usage in the 25 tandemly repeated Mastigamoeba heptads. The nonsynonymous substitution is indicated in boldface and underlined, and the resulting amino acid change is shown to the left of the heptad in boldface.

Figure 3

Consensus trees from parsimony, neighbor-joining, and maximum-likelihood phylogenetic analyses. Branch lengths are from maximum-likelihood analyses. (A) Tree based on an alignment of 24 RNA polymerase largest-subunit homologues. Bootstrap support values (parsimony, neighbor-joining, and maximum-likelihood) are shown at nodes relevant to the branching position of Mastigamoeba. (B) The consensus tree based on RPB1 sequences produced in the absence of outgroups.

Figure 4

Indicators for long branch attraction among RPB1 sequences. (A) The number of unique changes in each sequence at otherwise universally conserved sites as well as sites with a conserved ancestral character shared only with archaebacterial outgroups. G, Giardia; Tr, Trichomonas; Ty, Trypanosoma; M, Mastigamoeba; P, Plasmodium; R, red algae; C, other crown taxa. Averages are shown for red algae and for other crown eukaryotes. (B) The number of times randomly generated sequences were attracted to each taxon in 100 independent parsimony analyses.

Figure 5

Substitutions (boldface and underlined) in the most highly conserved polymerase largest-subunit motifs. Ac, Acanthamoeba; At, Arabidopsis; Dd, Dictyostelium; Gl, Giardia; Hh, Halobacterium; Hs, Homo; Mi, Mastigamoeba; Mt, Methanobacterium; Sa, Sulfolobus; Sc, Saccharomyces; Tb, Trypanosoma; Tv, Trichomonas; B1, RPB1; C1, RPC1.