The phylogeny and evolution of deoxyribonuclease II: an enzyme essential for lysosomal DNA degradation

Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the genomes of various organisms against known DNase II query sequences, in order to determine the likely point of origin of this enzyme among cellular life forms. Its complete absence from any other bacteria makes prokaryotic origin unlikely. Convincing evidence exists for DNase II homologs in Alveolates such as Paramecium, Heterokonts such as diatoms and water molds, and even tentative matches in green algae. Apparent absences include red algae, plants, fungi, and a number of parasitic organisms. Based on this phylogenetic distribution and hypotheses of eukaryotic relationships, the most probable explanation is that DNase II has been subject to multiple losses. The point of origin is debatable, though its presence in Trichomonas and perhaps in other evolutionarily basal "Excavate" protists such as Reclinomonas, strongly support the hypothesis that DNase II arose as a plesiomorphic trait in eukaryotes. It probably evolved together with phagocytosis, specifically to facilitate DNA degradation and bacteriotrophy. The various absences in many eukaryotic lineages are accounted for by loss of phagotrophic function in intracellular parasites, in obligate autotrophs, and in saprophytes.     

Does complexity constrain organelle evolution?

The evolution of eukaryotes was punctuated by invasions of the bacteria that have evolved to mitochondria and plastids. These bacterial endosymbionts founded major eukaryotic lineages by enabling them to carry out aerobic respiration and oxygenic photosynthesis. Yet, having evolved as free-living organisms, they were at first poorly adapted organelles. Although mitochondria and plastids have integrated within the physiology of eukaryotic cells, this integration has probably been constrained by the high level of complexity of their bacterial ancestors and the inability of gradual evolutionary processes to drastically alter complex systems. Here, I review complex processes that directly involve translation of plastid mRNAs and how they could constrain transfer to the nucleus of the genes encoding them.     

Origin and evolution of spliceosomal introns

ABSTRACT: Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded 'introns first' held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers' Reports section.     

The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells.

Eukaryotes have long been thought to have arisen by evolving a nucleus, endomembrane, and cytoskeleton. In contrast, it was recently proposed that the first complex cells, which were actually proto-eukaryotes, arose simultaneously with the acquisition of mitochondria. This so-called symbiotic association hypothesis states that eukaryotes emerged when some ancient anaerobic archaebacteria (hosts) engulfed respiring alpha-proteobacteria (symbionts), which evolved into the first energy-producing organelles. Therefore, the intracellular compartmentalization of the energy-converting metabolism that was bound originally to the plasma membrane appears to be the key innovation towards eukaryotic genome and cellular organization. The novel energy metabolism made it possible for the nucleotide synthetic apparatus of cells to be no longer limited by subsaturation with substrates and catalytic components. As a consequence, a considerable increase has occurred in the size and complexity of eukaryotic genomes, providing the genetic basis for most of the further evolutionary changes in cellular complexity. On the other hand, the active uptake of exogenous DNA, which is general in bacteria, was no longer essential in the genome organization of eukaryotes. The mitochondrion-driven scenario for the first eukaryotes explains the chimera-like composition of eukaryotic genomes as well as the metabolic and cellular organization of eukaryotes.     

Sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus

The origin of the eukaryotic cell cycle, including mitosis, meiosis, and sex are as yet unresolved aspects of the evolution of the eukaryotes. The wide phylogenetic distribution of both mitosis and meiosis suggest that these processes are integrally related to the origin of the earliest eukaryotic cells. According to the viral eukaryogenesis (VE) hypothesis, the eukaryotes are a composite of three phylogenetically unrelated organisms: a viral lysogen that evolved into the nucleus, an archaeal cell that evolved into the eukaryotic cytoplasm, and an alpha-proteobacterium that evolved into the mitochondria. In the extended VE hypothesis presented here, the eukaryotic cell cycle arises as a consequence of the derivation of the nucleus from a lysogenic DNA virus.     

Reconstruction of ancestral protosplice sites

Most of the eukaryotic protein-coding genes are interrupted by multiple introns. A substantial fraction of introns occupy the same position in orthologous genes from distant eukaryotes, such as plants and animals, and consequently are inferred to have been inherited from the common ancestor of these organisms. In contrast to these conserved introns, many other introns appear to have been gained during evolution of each major eukaryotic lineage. The mechanism(s) of insertion of new introns into genes remains unknown. Because the nucleotides that flank splice junctions are nonrandom, it has been proposed that introns are preferentially inserted into specific target sequences termed protosplice sites. However, it remains unclear whether the consensus nucleotides flanking the splice junctions are remnants of the original protosplice sites or if they evolved convergently after intron insertion. Here, we directly address the existence of protosplice sites by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and accordingly are not subject to selection for splicing efficiency. We show that introns are either predominantly inserted into specific protosplice sites, which have the consensus sequence (A/C)AG/Gt, or that they are inserted randomly but are preferentially fixed at such sites.     

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts by Legionella pneumophila

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF‐κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC–Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.     

Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> involved in chitin degradation in the bulb mite <Emphasis Type="Italic">Rhizoglyphus robini</Emphasis>

Cymbomonas tetramitiformis is a peculiar green alga that unites in one cell the abilities of photosynthesis and phagocytosis, which makes it a very useful model for the study of the evolution of plastid endosymbiosis. We have pondered over this issue and propose an evolutionary scenario of trophic strategies in eukaryotes, including primary and secondary plastid endosymbioses. C. tetramitiformis is a prototroph, just like the common ancestor of Archaeplastida was, and can synthesize most small organic molecules contrary to other eukaryotic phagotrophs, e.g. some metazoans, amoebozoans, and ciliates, which have not evolved tight endosymbiotic relationships. In order to establish a permanent photosynthetic endosymbiont they do not have to become prototrophs, but have to acquire the genes necessary for plastid retention via horizontal (including endosymbiotic) gene transfer. Such processes occurred successfully in the ancestors of eukaryotes with permanent secondary plastids and thus led to their great diversification. The preservation of phagocytosis in Cymbomonas (and some other prasinophytes as well) seems to result from nutrient deficiency in their oligotrophic habitats. This forces them to supplement their diet with phagocytized prey, in contrasts to the thecate amoeba Paulinella chromatophora, which also successfully transformed cyanobacteria into permanent organelles. Although Paulinella endosymbionts were acquired very recently in comparison to primary plastids, Paulinella has lost the ability to phagocytose, most probably due to the fact that it inhabits nutrient-rich environments, which renders the phagotrophy nonessential.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

Domestication of self-splicing introns during eukaryogenesis: the rise of the complex spliceosomal machinery

?

The spliceosome is a eukaryote-specific complex that is essential for the removal of introns from pre-mRNA. It consists of five small nuclear RNAs (snRNAs) and over a hundred proteins, making it one of the most complex molecular machineries. Most of this complexity has emerged during eukaryogenesis, a period that is characterised by a drastic increase in cellular and genomic complexity. Although not fully resolved, recent findings have started to shed some light on how and why the spliceosome originated.In this paper we review how the spliceosome has evolved and discuss its origin and subsequent evolution in light of different general hypotheses on the evolution of complexity. Comparative analyses have established that the catalytic core of this ribonucleoprotein (RNP) complex, as well as the spliceosomal introns, evolved from self-splicing group II introns. Most snRNAs evolved from intron fragments and the essential Prp8 protein originated from the protein that is encoded by group II introns. Proteins that functioned in other RNA processes were added to this core and extensive duplications of these proteins substantially increased the complexity of the spliceosome prior to the eukaryotic diversification. The splicing machinery became even more complex in animals and plants, yet was simplified in eukaryotes with streamlined genomes. Apparently, the spliceosome did not evolve its complexity gradually, but in rapid bursts, followed by stagnation or even simplification. We argue that although both adaptive and neutral evolution have been involved in the evolution of the spliceosome, especially the latter was responsible for the emergence of an enormously complex eukaryotic splicing machinery from simple self-splicing sequences.

Reviewers

This article was reviewed by W. Ford Doolittle, Eugene V. Koonin and Vivek Anantharaman.

     

Changing perspectives on the origin of eukaryotes

From the initial application of molecular techniques to the study of microbial organisms, three domains of life emerged, with eukaryotes and archaea as sister taxa. However, recent analyses of an expanding molecular data set reveal that the eukaryotic genome is chimeric with respect to archaea and bacteria. Moreover, there is now evidence that the primitive eukaryotic group ‘Archezoa' once harbored mitochondia. These discoveries have challenged the traditional stepwise model of the evolution of eukaryotes, in which the nucleus and microtubules evolve before the acquisition of mitochondria, and consequently compel a revision of existing models of the origin of eukaryotic cells.     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.     

Ancestral paralogs and pseudoparalogs and their role in the emergence of the eukaryotic cell

Gene duplication is a crucial mechanism of evolutionary innovation. A substantial fraction of eukaryotic genomes consists of paralogous gene families. We assess the extent of ancestral paralogy, which dates back to the last common ancestor of all eukaryotes, and examine the origins of the ancestral paralogs and their potential roles in the emergence of the eukaryotic cell complexity. A parsimonious reconstruction of ancestral gene repertoires shows that 4137 orthologous gene sets in the last eukaryotic common ancestor (LECA) map back to 2150 orthologous sets in the hypothetical first eukaryotic common ancestor (FECA) [paralogy quotient (PQ) of 1.92]. Analogous reconstructions show significantly lower levels of paralogy in prokaryotes, 1.19 for archaea and 1.25 for bacteria. The only functional class of eukaryotic proteins with a significant excess of paralogous clusters over the mean includes molecular chaperones and proteins with related functions. Almost all genes in this category underwent multiple duplications during early eukaryotic evolution. In structural terms, the most prominent sets of paralogs are superstructure-forming proteins with repetitive domains, such as WD-40 and TPR. In addition to the true ancestral paralogs which evolved via duplication at the onset of eukaryotic evolution, numerous pseudoparalogs were detected, i.e. homologous genes that apparently were acquired by early eukaryotes via different routes, including horizontal gene transfer (HGT) from diverse bacteria. The results of this study demonstrate a major increase in the level of gene paralogy as a hallmark of the early evolution of eukaryotes.     

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.     

The cellular machineries responsible for the division of endosymbiotic organelles

Chloroplasts (plastids) and mitochondria evolved from endosymbiotic bacteria. These organelles perform vital functions in photosynthetic eukaryotes, such as harvesting and converting energy for use in biological processes. Consistent with their evolutionary origins, plastids and mitochondria proliferate by the binary fission of pre-existing organelles. Here, I review the structures and functions of the supramolecular machineries driving plastid and mitochondrial division, which were discovered and first studied in the primitive red alga Cyanidioschyzon merolae. In the past decade, intact division machineries have been isolated from plastids and mitochondria and examined to investigate their underlying structure and molecular mechanisms. A series of studies has elucidated how these division machineries assemble and transform during the fission of these organelles, and which of the component proteins generate the motive force for their contraction. Plastid- and mitochondrial-division machineries have important similarities in their structures and mechanisms despite sharing no component proteins, implying that these division machineries evolved in parallel. The establishment of these division machineries might have enabled the host eukaryotic ancestor to permanently retain these endosymbiotic organelles by regulating their binary fission and the equal distribution of resources to daughter cells. These findings provide key insights into the establishment of endosymbiotic organelles and have opened new avenues of research into their evolution and mechanisms of proliferation.     

Was the initiation of translation in early eukaryotes IRES-driven?

The initiation of translation in eukaryotes generally involves the recognition of a 'cap' structure at the 5' end of the mRNA. However, for some viral and cellular mRNAs, a cap-independent mechanism occurs through an mRNA structure known as the internal ribosome entry site (IRES). Here, I postulate that the first eukaryotic mRNAs were translated in a cap-independent, IRES-driven manner that was then superseded in evolution by the cap-dependent mechanism, rather than vice versa. This hypothesis is supported by the following observations: (i) IRES-dependent, but not cap-dependent, translation can take place in the absence of not only a cap, but also many initiation factors; (ii) eukaryotic initiation factor 4E (eIF4E) and eIF4G, molecules absolutely required for cap-dependent translation, are among the most recently evolved translation factors; and (iii) functional similarities suggest the evolution of IRESs from spliceosomal introns. Thus, the contemporary cellular IRESs might be relics of the past.     

Parallels in genome evolution in mitochondria and bacterial symbionts

Mitochondria, the energy-producing organelles of the eukaryotic cell, originate from an endosymbiotic alpha-proteobacterium. These organelles are believed to have arisen only once in evolutionary history, but despite their common ancestry, mitochondrial DNAs vary extensively throughout eukaryotes in genome architecture and gene content. New insights into early mitochondrial genome evolution come from the investigation of primitive mitochondriate eukaryotes, as well as the comparison between mitochondria and intracellular bacterial symbionts.