Here, we describe, in several amoebozoan genera (Dictyostelium, Hartmannella, and Physarum), experimentally confirmed mtDNA-encoded RNA species that have several of the generic characteristics of 5S rRNA, namely, high abundance, small size (97 to ~130 nt), ability to adopt a 5S rRNA-like secondary structure, and (in the two cases investigated here) association with mitoribosomes in ultracentrifugation experiments. Moreover, we have identified in silico what appear to be homologous sequences in the mitochondrial genome of several additional Dictyostelium species and in a closely related slime mold genus, Polysphondylium, as well as in a more distantly related myxomycete slime mold, D. nigripes. We predict that these sequences are also expressed as small abundant RNAs in the mitochondria of these other amoebozoan taxa. No conserved open reading frames (ORFs) and no AUG-initiated ORFs of >20 codons in length are contained within these small RNAs or their putative homologs.
Within the two relatively closely related genera Dictyostelium and Polysphondylium, sequence and positional conservation of the genes in question is evident although sequence identity is restricted to relatively short stretches. Thus, even where there is reasonable evidence of homology, the sequences per se are highly divergent. The same situation applies to Physarum and Didymium. In fact, pairwise comparisons demonstrate that the putative mitochondrial 5S rRNA sequences are evolving at a considerably more rapid rate than the corresponding LSU rRNA sequences in the same organisms, with the former consistently exhibiting identity values 10 to 30% lower than the latter. For example, for D. discoideum versus D. citrinum, ddiRNA and dciRNA sequences are only 83.7% identical, whereas the corresponding LSU rRNA sequences (disregarding introns) share 94.0% identity. Very similar values (82.7% for ppaRNA; 94.0% for LSU rRNA) are observed with P. pallidum CK8 versus P. pallidum PN500, whereas a wider spread is seen in a D. fasciculatum-P. pallidum CK8 comparison (86.0% identity for LSU rRNA and 57.8% identity for ddiRNA versus ppaRNA-CK8). In a broader comparison involving Dictyostelium/Polysphondylium, Hartmannella, and Physarum/Didymium, there is no compelling sequence-based evidence of homology among all of the small RNAs described here. Nevertheless, considering that in three instances these sequences are known to be expressed as abundant small RNAs—and in the case of Physarum to undergo the type of insertional editing that characterizes maturation of mitochondrial mRNA, rRNA, and tRNA transcripts in this organism—it is highly likely that these small abundant RNAs are functional in the mitochondria of the organisms in which they are present.
In the absence of both a functional test for 5S rRNA and in vitro
mitochondrial translation systems for the amoebozoan taxa investigated here, we cannot definitively assert that these small RNAs are functional counterparts of the 5S rRNA found in other mitoribosomes; however, several of their properties support this hypothesis. Cofractionation of the small RNAs identified here with mitoribosomes (e.g., in ultracentrifugation experiments) is perhaps the strongest indication that these RNAs may play a role in ribosome function. In this respect, it is noteworthy that Pi et al. (24
) found that in their ultracentrifugation experiments, the ddiRNA (msRNA in their study) was localized almost exclusively in the high-speed supernatant rather than in the mitoribosome-containing pellet. However, it is likely that dissociation of ribosome-bound ddiRNA was promoted by the relatively high ionic conditions the authors used (buffer containing 250 mM KCl).
In two cases (Hartmannella and Physarum), isolated mitochondrial RNA contained a species identified as cytoplasmic 5S rRNA in addition to the small mtDNA-encoded RNAs characterized here. However, it is unlikely that a nuclear DNA-encoded 5S rRNA is imported into mitochondria to replace the function of a mtDNA-encoded 5S rRNA in these taxa because in ultracentrifugation experiments, Hartmannella cytoplasmic 5S rRNA did not cofractionate with mitoribosomes, whereas hveRNA did.
Computational modeling corroborates the notion that the amoebozoan small RNAs structurally resemble 5S rRNAs, but whether they are derived homologs or analogs originating from convergent evolution cannot be distinguished by the approach we employed.
In view of the extraordinary structural plasticity displayed by mitoribosomes (20
) and in marked contrast to the structural conservatism of their bacterial counterparts, it is not unreasonable to suppose that a conventional 5S rRNA might be replaced by some other molecule (RNA or protein) in the mitoribosome of certain organisms. In this regard, we note that in the large subunit of the mammalian mitoribosome, which lacks a 5S rRNA component, the region normally occupied by 5S rRNA (the so-called central protuberance) is, in fact, replaced by protein. Atomic resolution three-dimensional structures suggest that a protein element termed the LSU handle may assume some of the roles of 5S rRNA in the mitoribosome (28
). Thus, molecular mimicry, in which a nonhomologous 5S rRNA-like analog is able to structurally and functionally substitute for a conventional 5S rRNA, is a possibility that we cannot entirely discount. However, while there is precedent for substitution of a protein for RNA in mitoribosomes, there is none for substitution of RNA for RNA. Hence, if the mitochondrial RNA species described here do function as 5S rRNA counterparts, in all probability they are 5S rRNA homologs (albeit highly divergent) rather than analogs. The view that a 5S rRNA was encoded in the mtDNA of the common ancestor of Amoebozoa and subsequently diverged through speciation is further supported by the presence of a recognizable mitochondrial 5S rRNA in A. castellanii
. By extrapolation, highly derived (but currently unrecognized) 5S rRNAs may also exist in other mitochondrial systems that appear to lack a conventional 5S rRNA.
Definitive proof of the hypothesis advanced here will require a demonstration that a 5S rRNA-like counterpart is not only a component of the large mitoribosomal subunit in one or more of the organisms studied here but also that it occupies the analogous position within the ribosome's three-dimensional structure. In turn, this will require the isolation and crystallization of a sufficient quantity of pure amoebozoan mitoribosomes to allow determination of the tertiary structure.