A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes

gb-2004-5-2-r7 GBJ Research A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes Koonin V Eugene koonin@ncbi.nlm.nih.gov Fedorova D Natalie Jackson D John Jacobs R Aviva Krylov M Dmitri Makarova S Kira Mazumder Raja Mekhedov L Sergei Nikolskaya N Anastasia Rao Sridhar B Rogozin B Igor Smirnov Sergei Sorokin V Alexander Sverdlov V Alexander Vasudevan Sona Wolf I Yuri Yin J Jodie Natale A Darren

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA

Current address: Protein Identification Resource, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007, USA

Genome Biology 1465-6906 2004 5 2 R7 http://genomebiology.com/2004/5/2/R7 14759257 23 10 2003 1 12 2003 4 12 2003 15 1 2004 2004 Koonin et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs from seven eukaryotic genomes. The analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes.

Abstract

Background

Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes.

Results

We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi. Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability. The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing. These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes. The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail. For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context. Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number. Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution. Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes. Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed. The gene set of the last common ancestor of the crown group consists of 3,413 KOGs and largely includes proteins involved in genome replication and expression, and central metabolism. Only 44% of the KOGs, mostly from the reconstructed gene set of the last common ancestor of the crown group, have detectable homologs in prokaryotes; the remainder apparently evolved via duplication with divergence and invention of new genes.

Conclusions

The KOG analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes. The results provide quantitative support for major trends of eukaryotic evolution noticed previously at the qualitative level and a basis for detailed reconstruction of evolution of eukaryotic genomes and biology of ancestral forms.

Evolution Genome studies Bioinformatics Model organisms

Background

Comparative analysis of genomes from distant species provides new insights into gene functions, genome evolution and phylogeny. In particular, the comparative genomics of prokaryotes has revealed previously underappreciated major trends in genome evolution, namely, extensive lineage-specific gene loss and horizontal gene transfer (HGT) 1234567. To efficiently extract functional and evolutionary information from multiple genomes, rational classification of genes based on homologous relationships is indispensable. The two principal classes of homologs are orthologs and paralogs 891011. Orthologs are defined as homologous genes that evolved via vertical descent from a single ancestral gene in the last common ancestor of the compared species. Paralogs are homologous genes, which, at some stage of evolution, have evolved by duplication of an ancestral gene. Orthology and paralogy are intimately linked because, if a duplication (or a series of duplications) occurs after the speciation event that separated the compared species, orthology becomes a relationship between sets of paralogs, rather than individual genes (in which case, such genes are called co-orthologs).

Correct identification of orthologs and paralogs is of central importance for both the functional and evolutionary aspects of comparative genomics 1213. Orthologs typically occupy the same functional niche in different organisms; in contrast, paralogs evolve to functional diversification as they diverge after the duplication 141516. Therefore, robustness of genome annotation depends on accurate identification of orthologs. A clear demarcation of orthologs and paralogs is also required for constructing evolutionary scenarios, which include, along with vertical inheritance, lineage-specific gene loss and HGT 57.

In principle, orthologs, including co-orthologs, should be identified by means of phylogenetic analysis of entire families of homologous proteins, which is expected to define orthologous protein sets as clades 171819. However, for genome-wide protein sets, such analysis remains extremely labor-intensive, and error-prone as well. Accordingly, procedures have been developed for identifying sets of likely orthologs without explicit referral to phylogenetic analysis. These procedures are based on the notion of a genome-specific best hit (BeT), that is, the protein from a target genome that is most similar (typically in terms of similarity scores computed using BLAST or another sequence-comparison method) to a given protein from the query genome 2021. The assumption central to this approach is that orthologs have a greater similarity to each other than to any other protein from the respective genomes. When multiple genomes are analyzed, pairs of probable orthologs detected on the basis of BeTs are combined into orthologous clusters represented in all or a subset of the analyzed genomes 2022. This approach, amended with additional procedures for detecting co-orthologous protein sets and for treating multidomain proteins, was implemented in the database of Clusters of Orthologous Groups (COGs) of proteins 202324. The current COG set includes approximately 70% of the proteins encoded in 69 genomes of prokaryotes and unicellular eukaryotes 25. The COGs have been used for functional annotation of new genomes 26272829, target selection in structural genomics 303132, identification of potential drug targets 3334 and genome-wide evolutionary studies 41335363738. Sonnhammer and co-workers independently developed a similar methodology for identification of co-orthologous protein sets from pairwise genome comparisons and applied it to the sequenced eukaryotic genomes 39.

A central notion introduced in the context of the COG analysis is that of a phyletic pattern, that is, the pattern of representation (presence-absence) of analyzed species in each COG 1320. Similar concepts have been independently developed and applied by others 4041. The COGs show a remarkable scatter of phyletic patterns, with only a small minority represented in all sequenced genomes. A recent quantitative study showed that parsimonious evolutionary scenarios for most COGs involve multiple events of gene loss and HGT 7. Both similarity and complementarity among the phyletic patterns of COGs, in conjunction with other information, such as conservation of gene order, have been successfully employed to predict gene functions 134243. The comparison of phyletic pattern has been formalized in set-theoretical algorithms and systematically applied to the computational and experimental analysis of bacterial flagellar systems, which demonstrated the considerable robustness of this approach 44.

We recently extended the system of orthologous protein clusters to complex, multicellular eukaryotes 25. Here, we examine the phyletic patterns of KOGs in connection with known and predicted protein functions. In-depth analysis of some of these KOGs resulted in prediction of previously uncharacterized, but apparently essential, conserved eukaryotic protein functions. We also reconstruct the parsimonious scenario of evolution of the crown-group eukaryotes by assigning the loss of genes (KOGs) and emergence of new genes to the branches of the phylogenetic tree and explicitly delineate the minimal gene sets for various ancestral forms. To our knowledge, this is the first systematic, genome-wide examination of the sets of orthologous genes in eukaryotes.

Results and discussion

KOGs for seven sequenced eukaryotic genomes: functional and evolutionary implications of phyletic patterns

Eukaryotic KOGs were constructed on the basis of the comparison of proteins encoded in the genomes of three animals (Homo sapiens 45, the fruit fly Drosophila melanogaster 46 and the nematode Caenorhabditis elegans 47), the green plant Arabidopsis thaliana (thale cress) 48, two fungi (budding yeast Saccharomyces cerevisiae 49 and fission yeast Schizosaccharomyces pombe 50) and the microsporidian Encephalitozoon cuniculi 51. The procedure for KOG construction was a modification of the one previously used for COGs 2024 and is described in greater detail elsewhere (25; see also Materials and methods). An important difference stems from the fact that complex eukaryotes encode many more multidomain proteins than prokaryotes and, furthermore, orthologous eukaryotic proteins often differ in domain composition, with additional domains accrued in more complex forms 345. Accordingly, and unlike the original COG construction procedure, probable orthologs with different domain architectures were assigned to one KOG and were not split if they shared a common core of domains. In addition to the KOGs, which consisted of at least three species, clusters of putative orthologs from two species (TWOGs) and lineage-specific expansions (LSEs) of paralogs from each of the analyzed genomes were identified (2552; see also Materials and methods). In most of the analyses discussed below, KOGs and TWOGs are treated together, unless otherwise specified.

Figure 1 shows the assignment of the proteins from each of the analyzed eukaryotes to KOGs with different numbers of species, TWOGs and LSEs. The fraction of proteins assigned to KOGs tends to decrease with the increasing genome size, from 81% for S. pombe to 51% for the largest, the human genome. (For reasons that remain unclear, but might be related to its intracellular parasitic lifestyle, E. cuniculi has a relatively small fraction of conserved proteins that belonged to KOGs: approximately 60%.) The contribution of LSEs shows the opposite trend, being the greatest in the largest genomes, that is, human and Arabidopsis, and minimal in the microsporidian (Figure 1). A notable difference was observed between eukaryotes in terms of their representation in KOGs found in different numbers of species. While the three unicellular organisms are represented mainly in the highly conserved seven- or six-species KOGs, a much larger fraction of the gene set in animals and Arabidopsis is accounted for by LSEs, and by KOGs found in three or four genomes. These include animal-specific genes and genes that are shared by plants and animals but not by fungi and the microsporidian (Figure 1). The large number of KOGs in the latter group (700 KOGs represented in Arabidopsis and at least two animal species) is notable and probably results from massive, lineage-specific loss of genes during eukaryotic evolution (see below).

Figure 1

Assignment of proteins from each of the seven analyzed eukaryotic genomes to KOGs with different numbers of species and to LSEs

Assignment of proteins from each of the seven analyzed eukaryotic genomes to KOGs with different numbers of species and to LSEs. 0, Proteins without detectable homologs (singletons); 1, LSEs. Species abbreviations: Ath, Arabidopsis thaliana; Cel, Caenorhabditis elegans; Dme, Drosophila melanogaster; Ecu, Encephalitozoon cuniculi; Hsa, Homo sapiens; Sce, Saccharomyces cerevisisae; Spo, Schizosaccharomyces pombe.

The phyletic patterns of KOGs reveal both the existence of a conserved eukaryotic gene core and substantial diversity. The 'pan-eukaryotic' genes, which are represented in each of the seven analyzed genomes, account for around 20% of the KOGs, and approximately the same number of KOGs include all species except for the microsporidian, an intracellular parasite with a highly degraded genome 51. Among the remaining KOGs, a large group includes representatives of the three analyzed animal species (worm, fly and humans) but a substantial fraction (approximately 30%) are KOGs with unexpected patterns, for example, one animal, one plant and one fungal species (see 53 and examples in Table 1).

Table 1

KOGs and TWOGs with unexpected phyletic patterns (examples)

KOG/TWOG number

Phyletic pattern*

(Predicted) structure and function

Prokaryotic homologs

Comments

TWOG0892

---H--E

Discoidin domain protein, potential regulator of proteasome activity

Detected in a few phylogenetically scattered bacteria, no COG so far 69

TWOG0263

A-----E

ATP/ADP translocase

ATP/ADP translocases of chlamydia, rickettsia, Xylella fastidiosa

ATP/ADP translocase is a hallmark of intracellular parasites and symbionts, which allows them to scavenge ATP from the host cell; chloroplast protein in plants. Could be acquired by plants and microsporidia via independent HGT from bacteria. 58

TWOG0689

---HY--

Uncharacterized protein essential for propionate metabolism

PrpD protein of several bacteria and archaea (COG2079)

The yeast and human (and the orthologs from other vertebrates) proteins show the greatest similarity to different subsets of bacterial orthologs, which might suggest independent HGT events.

TWOG0871

---H-P-

Uncharacterized conserved protein, probably enzyme

COG4336, sporadic representation in several bacterial lineages

The human (and mouse) protein has an additional domain conserved in the archaeon Pyrococcus. Human and S. pombe proteins are most similar to different subsets of bacterial homologs, which suggests the possibility of independent HGT events.

TWOG0788

A----P-

Urease

Ureases of many bacterial species

Highly conserved enzyme present in plants and many fungi but not S. cerevisiae. Plant and fungal ureases have a common domain architecture distinct from that of bacterial orthologs, which suggests monophyletic origin. Might have evolved via early HGT from bacteria (proto-mitochondria?) with subsequent loss in animals and some fungi.

4751

A--H--E

Recombination repair protein BRCA2, contains varying number of BRCA2 repeats

None

Although sequence conservation is limited to the BRC repeats 101 the number of which varies substantially, statistical significance of the observed sequence similarity and the absence of other homologs suggests that the proteins in this KOG are true orthologs. Apparent orthologs of BRCA2 are detectable also in other species from the taxa represented in the KOGs (mosquito Anopheles gambiae, fungus Ustilago maydis) 102 and in early-branching eukaryotes (Leishmania, Trypanosoma; E.V.K., unpublished work), suggesting that evolution of BRCA2 involved multiple gene losses

4597

A--H--E

TATA-binding protein 1-interacting protein

None

Probable multiple gene losses

4486

A--H--E

3-methyl-adenine DNA glycosylase

Orthologs in many bacteria (COG2094)

The plant protein and those from mammals and microsporidia show the greatest similarity to different subsets of bacterial orthologs. Evolution might have included a combination of gene loss and independent HGT events

1594

A-D-Y--

Predicted epimerase related to aldose 1-epimerase

Bacterial orthologs, primarily proteobacteria (COG0676)

Eukaryotic proteins are more closely related to each other than to bacterial orthologs, indicating monophyletic origin. Function remains unknown; might be involved in a distinct and still uncharacterized pathway of polysaccharide biosynthesis. LSE in Arabidopsis (seven paralogs).

4141

---HYPE

Rad52/22, protein involved in double-strand break repair

None

Probable gene loss in plants, insects and nematodes

4528

-CDH--E

Uncharacterized predicted enzyme, possibly a polynucleotide kinase (structure of the ortholog from the bacterium Thermotoga maritima has been determined - pdb code 1j5u)

Conserved in all archaea and several bacteria (COG1371)

Context analysis of archaeal and bacterial genomes suggests functional interaction between proteins of KOG5324 and KOG4246, RNA 3'-terminal phosphate cyclase (KOG4398, COG0430), and tRNA/rRNA cytosine C5-methylase (KOG1299/COG0144) (103 and E.V.K., unpublished observations). Taken together, the observations appear to implicate KOG5324 and KOG4246 in a still uncharacterized pathway of rRNA and/or tRNA processing and modification. Conservation of these proteins in archaea and early-branching eukaryotes suggests lineage-specific gene loss in plants and fungi.

3833

-CDH--E

Uncharacterized predicted enzyme, possibly a polynuclotide phosphatase

Conserved in all archaea and several bacteria (COG1690)

See comment for KOG5324

*Abbreviations: A, thale cress A. thaliana; C, nematode C. elegans; D, fruit fly D. melanogaster; E, microsporidian Encephalitozoon cuniculi; H, Homo sapiens; S, budding yeast S. cerevisiae; P, fission yeast S. pombe; a letter indicates the presence of the respective species in the given KOG and a dash indicates its absence.

During the manual curation of the KOG set, the KOGs with unexpected patterns were scrutinized in an effort to detect potential highly diverged members from one or more of the analyzed genomes. Some of these unexpected patterns might indicate that a gene is still missing in the analyzed set of protein sequences from one or more of the species included; reports of newly discovered genes have appeared since the release of the initial reports on genome sequences of complex eukaryotes, for example, as a result of massive sequencing of human cDNAs 54, exhaustive annotation of the Drosophila genome 55 and comparative analysis of closely related yeast genomes 56. The unexpected phyletic patterns seem, however, largely to reflect the extensive, lineage-specific gene loss that is characteristic of eukaryotic evolution 57; on many occasions, this scenario is supported by the presence of orthologs in other eukaryotic lineages and/or in prokaryotes (Table 1). However, interesting exceptions to the multiple loss explanation might exist as exemplified by the ATP/ADP-translocase, which is present in Arabidopsis and Encephalitozoon and could have evolved via independent HGT from intracellular bacterial parasites (58 and Table 2).

Table 2

KOGs represented by exactly one ortholog in seven analyzed eukaryotic genomes (examples)

KOG number

(Predicted) function

Multiprotein complex

Functional class*

Prokaryotic homologs

Fitness class^†

Comments

Yeast^‡

Worm^§

Genes experimentally or computationally characterized previously

0392

SNF2 family DNA-dependent ATPase

TBP-DNA complex

Many bacteria and archaea (COG0553)

Involved in regulation of transcription from POL II promoters 104

0121

Nuclear cap-binding protein complex, subunit CBP20 (RRM-domain-containing RNA-binding protein)

Cap-binding complex

Several bacteria (COG0724)

RRM-domain proteins show scattered presence in bacteria and might have been horizontally transferred from eukaryotes

0213

U2-snRNP associated splicing factor 3b, subunit 1

Spliceosome

None

0227

snRNA-associated protein, splicing factor 3a, subunit b (Prp11p)

Spliceosome

None

2268

Predicted nucleic-acid-binding protein kinase of the RIO1 family; 40S ribosomal subunit biogenesis/18S rRNA processing

Pre-40S subunit

Orthologs in most archaea but not in bacteria (COG0478)

One of the very small number of protein kinases that show a clear-cut orthologous relationship between all eukaryotes and most archaea, and, apparently, the only one containing a helix-turn-helix nucleic-acid-binding domain. 105 Associated with yeast pre-40S subunit and required for its maturation. 106

3031

Protein required for 60S ribosomal subunit biogenesis; 107 contains the IMP4 domain, which is involved in rRNA processing 108; paralog of KOG3095 and KOG3292, which are also represented in all analyzed genomes.

Processosome

Distantly related to COG2136, represented by orthologs in most archaea, but not in bacteria (KSM, unpublished)

The COG2136 proteins appear to be subunits of the predicted archaeal exosome 109. Apparently, this gene has undergone at least two ancient duplications in eukaryotes

3045

Predicted RNA methylase involved in rRNA processing

Processosome?

Distantly related to numerous Rossmann-fold methylases but prokaryotic orthologs could not be confidently identified

This protein (Rrp8p in yeast) has been shown to participate in the processing of rRNA and sequence analysis reveals the presence of a Rossmann-fold methylase domain 110. Therefore Rrp8p probably methylates either snoRNA or rRNA itself.

3064

RNA-binding nuclear protein containing a distinct C4 Zn-finger; implicated in the biogenesis of 60S ribosomal subunits 111

Processosome

None

Initially identified in yeast as the MAK16 protein required for dsRNA virus reproduction 112

0291, 0302, 0306, 310, 0319, 0650, 1272

WD40-repeat proteins, subunits of rRNA processing complexes 6970

Processosome

WD40-repeat proteins are present in several bacterial lineages and are particularly abundant in cyanobacteria but are missing in most archaea; none of them appear to be obvious orthologs of this protein (COG2319)

all 0

X,X,1,X,1,1,1

0284

Polyadenylation factor I complex, subunit PFS2, WD40-repeat protein

Poly-adenylation complex

Same as above (COG2319)

0337

RNA helicase involved in 28S rRNA processing

Processosome

Most of the archaea and bacteria (COG0513)

0343

RNA helicase involved in 28S rRNA processing

Processosome

Most of the archaea and bacteria (COG0513)

1069

3'-5' exoribonuclease (RNAse PH), exosome subunit Rrp46

Exosome

Most bacteria and archaea (COG0689)

1070

Exosome subunit Rrp5 (RNA-binding S1 domain fused to TPR repeats)

Exosome

Most bacteria (COG0539, COG0457)

1135

mRNA cleavage and polyadenylation complex subunit CFT2 (CPSF)

Cleavage and polyadenylation complex

Most archaea and some bacteria (COG1236)

1914

mRNA cleavage and polyadenylation factor I complex, subunit RNA14

Cleavage and polyadenylation complex

None

1975

RNA (guanine-7-) methyltransferase (capping enzyme subunit)

Capping enzyme

Numerous methyltrans-ferases (COG0500) but no ortholog

2051

Nonsense-mediated mRNA decay complex, subunit 2

NMD complex

None

2554

Pseudouridylate synthase

Most archaea and bacteria (COG0101)

2613

Upf1p-interacting protein, NMD complex subunit Nmd3p

NMD complex

All archaea, no bacteria (COG1499)

2771

tRNA-specific adenosine-34 deaminase subunit Tad3p

Heterodimeric RNA-specific deaminase

Most bacteria and some archaea (COG0590)

2780

Protein involved in ribosomal large subunit assembly (RPF1), contains IMP4 domain

Processosome

Most archaea, no bacteria (COG2136)

2781

Subunit of the small (ribosomal) subunit (SSU) processosome (snoRNP), IMP4

Processosome

Most archaea, no bacteria (COG2136)

2874

Protein involved in rRNA processing and ribosomal assembly

All archaea, no bacteria (COG1094)

Predicted RNA-binding protein containing KH domain

3013

Exosome subunit Rrp4

Exosome

Most archaea, on bacteria (COG1097)

3031

Protein involved in large ribosome subunit assembly and 28S rRNA processing (Rrf2)

Processosome

None

Contains the BRIX domain

3322

RNAse P/MRP subunit, involved in processing of pre-tRNAs and the 5.8S rRNA

RNAse P/MRP holoenzyme

None

3448

Predicted snRNP core protein

Spliceosome

All archaea, no bacteria (COG1958)

3482

Small nuclear ribonucleoprotein (snRNP) SMF subunit

Spliceosome

All archaea, no bacteria (COG1958)

2463

Predicted RNA-binding protein, consisting of a PIN domain and a Zn-ribbon. Involved in 26S proteasome assembly

26S proteasome, pre-40S subunit

A,O

Represented by orthologs in all archaea but no bacteria (COG1349)

PIN domain has been detected in exosome subunits and is thought to have RNA-binding properties or even nuclease activity 113114. The demonstration of the role of this protein (Nob1p) in proteasome assembly 115, 40S ribosome subunit assembly, and the processing of 18S rRNA 3'-end 116 supports the connection between degradation of RNA and proteins that seems to have been established already in archaea 109.

3273

Predicted RNA-binding protein containing KH domain, interacts with Nob1p

26S proteasome, pre-40S subunit

A,O

Orthologs in all archaea but no bacteria (COG1094)

This is the second predicted RNA-binding protein involved in proteasome assembly, 115 which emphasizes the aforementioned link between RNA and protein processing

1831

Deadenylating 3'-5' exonuclease, negative regulator of PolII transcription

CCR4-NOT core complex

None

1159

NADP-dependent flavoprotein reductase, probably sulfite reductase subunit

Many bacteria (COG0369)

Genetic evidence of a role in DNA replication 117

1800

Ferredoxin/adrenodoxin reductase

Most bacteria and some archaea (COG0493)

1173

Anaphase-promoting complex (APC), Cdc16 subunit (TPR-repeat protein)

APC

Most of archaea and bacteria have TPR-repeat proteins (COG0457) but no orthologs of Cdc16

3437

Anaphase-promoting complex (APC), subunit 10

APC

None

1358

Serine palmitoyltransferase

Most bacteria and some archaea (COG0156)

1511

Mevalonate kinase

Most archaea and some bacteria (COG1577)

3059

N-acetylglucosaminyltransferase complex, subunit PIG-C/GPI2, involved in phosphatidylinositol biosynthesis

N-acetylglucos-aminyltransferase complex

None

0467

Translation elongation factor 2 paralog (GTPase)

All (COG0480)

Involved in 60S ribosomal subunit maturation 118

1147

Glutamyl-tRNA synthetase

Multispecificity aminoacyl-tRNA synthetase complex

All (COG0008)

2784

Phenylalanyl-tRNA synthetase, beta subunit

Heterodimeric phenylalanyl-tRNA synthetase

All (COG0016)

3123

Diphtamide synthase (methyltransferase)

All archaea, no bacteria (COG1798)

0261

RNA polymerase III, largest subunit

RNAPIII holoenzyme

All (COG0086)

0262

RNA polymerase I, largest subunit

RNAPI holoenzyme

All (COG0086)

0215

RNA polymerase III, second largest subunit

RNAPIII holoenzyme

All (COG0085)

0216

RNA polymerase I, second largest subunit

RNAPI holoenzyme

All (COG0085)

1063

RNA polymerase II elongator complex, subunit ELP2, WD repeat protein

RNA polymerase II elongator complex

1131

RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, 5'-3' helicase subunit RAD3

RNAPII holoenzyme

Most archaea and bacteria (COG1199)

1920

RNA polymerase II Elongator subunit

RNAP II elongator complex

None

1932

TBP-associated factor (Taf2p)

TFIID complex

None

2009

Transcription initiation factor TFIIIB, Bdp1 subunit (Myb domain)

TFIIIB

None

2076

RNA polymerase III transcription factor TFIIIC, TPR-repeat-containing protein

TFIIIC

Most of archaea and bacteria have TPR-repeat proteins (COG0457) but no orthologs of TFIIC

2487

RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB4

TFIIH

None

2691

RNA polymerase II subunit 9

RNAP II holoenzyme

Most archaea, no bacteria (COG1594)

2807

RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, SSL1 subunit

TFIIH

No orthologs although von Willebrand A domains are present in a variety of prokaryotic proteins

Consists of a von Willebrand A domain most closely related to those in the proteasome subunit RPN10 119 and a Zn-finger domain

2907

RNA polymerase I transcription factor TFIIS, subunit A12.2/RPA12

TFIIS

All archaea, no bacteria (COG1594)

3169

RNA polymerase II transcriptional regulation mediator

Mediator complex 120

None

3233

RNA polymerase III subunit C34

RNAP III holoenzyme

None

3297

RNA polymerase III subunit C25

RNAP III holoenzyme

All archaea, no bacteria (COG1095)

3438

Subunit common to RNA polymerases I (A) and III (C); Rpc19p

RNAP I and III holoenzymes

3471

RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB2

TFIIH

None

3490

Transcription elongation factor SPT4, Zn-ribbon protein

Chromatin-associated transcription complexes

None

3497

RNA polymerase II subunit; Rpb10p

RNAP II holoenzyme

All archaea, no bacteria (COG1644)

3901

Transcription initiation factor IID subunit (Taf13p)

TFIID

None

3949

RNA polymerase II elongator complex, subunit ELP4

RNAP II elongator complex

None

4086

SOH1 protein potentially involved in Pol II transcription regulation and repair

SMCC complex 121

None

1532

Predicted GTPase of the XAB1 family 122

TBP-free TAF(II) complex

All archaea and several bacteria (COG1100)

XP-A-binding protein in humans, thus implicated in repair (122 and references therein).

1533

Predicted GTPase of the XAB1 family (paralog of KOG1757) 122

TBP-free TAF(II) complex?

All archaea and several bacteria (COG1100)

Might have a function in repair given the paralogous relationship with KOG1757.

1625

DNA polymerase α processivity subunit, inactivated phosphatase

DNA polymerase α holoenzyme

Small subunit of archaeal DNA polymerase II (COG1311)

The small, regulatory subunit of DNA polymerase α also forms a pan-eukaryotic KOG3044, which is a paralog of KOG0861 (the only recent duplication in KOG3044 is seen in vertebrates). In contrast, another paralog, the small subunit of DNA polymerase ε, is represented in animals, fungi and the early-branching protozoan Plasmodium, but not in plants or Microsporidia. Thus, the history of this polymerase subunit apparently involved inactivation of the phosphatase (or nuclease) inherited from archaea, with subsequent duplications at early stages of eukaryotic evolution 123

0479

DNA replication licensing factor MCM3

Pre-replication complex

All archaea, no bacteria (COG1241)

0481

DNA replication licensing factor MCM5

Pre-replication complex

All archaea, no bacteria (COG1241)

0482

DNA replication licensing factor MCM7

Pre-replication complex

All archaea, no bacteria (COG1241)

0964

Structural maintenance of chromosome protein 3 (cohesin subunit SMC3)

Sister chromatid cohesion complex

Many archaea and bacteria (COG1196)

0979

Structural maintenance of chromosome protein 5 (cohesin subunit SMC5)

Sister chromatid cohesion complex

Many archaea and bacteria (COG1196)

1942

TBP-interacting protein TIP49 (DNA helicase)

chromatin remodeling complex

Most of the archaea, no bacteria (COG1224)

1979

DNA mismatch repair ATPase, MLH1

Mismatch repair complex

Most bacteria and some archaea (COG0323)

2267

DNA primase, large subunit

DNA polymerase α:primase complex

All archaea, no bacteria (COG2219)

2299

Ribonuclease HI

Replisome

All archaea, most bacteria (COG0164)

2310

DNA repair exonuclease MRE11

MRN complex involved in double-strand break repair

All archaea, most bacteria (COG0420)

2929

Origin recognition complex, subunit 2 (ORC2)

ORC

None

0179

20S proteasome, regulatory subunit beta type PSMB1/PRE7 (paralog of KOG0185)

20S proteasome

All archaea but only actinomycetes among bacteria (COG0638)

0185

20S proteasome, regulatory subunit beta type PSMB4/PRE4 (paralog of KOG0179)

20S proteasome

All archaea but only actinomycetes among bacteria (COG0638)

2708

Predicted metalloprotease with chaperone activity (RNAse H/HSP70 fold) 124

Putative complex involved in translation regulation 125

Represented by orthologs in all archaea and bacteria (COG0533)

One of the few remaining uncharacterized proteins that are universally conserved in all cellular life forms. The only experimentally demonstrated activity is that of sialoglycoprotease but fusion with a distinct protein kinase in several archaea and analysis of gene neighborhood suggest a fundamental role in signal transduction, possibly translation regulation. 125

0301

Protein required for normal rates of ubiquitin-dependent proteolysis, contains WD40 repeats

Proteasome?

Same as above (COG2319)

0358

Chaperonin complex component, TCP-1 delta subunit (CCT4)

TCP-1

All archaea and nearly all bacteria (COG0459)

0363

Chaperonin complex component, TCP-1 beta subunit (CCT2)

TCP-1

All archaea and nearly all bacteria (COG0459)

0687

26S proteasome regulatory complex, subunit RPN7/PSMD6

26S proteasome

None

1299

Vacuolar sorting protein VPS45/Stt10 (Sec1 family)

t-SNARE complex

None

Involved in t-SNARE complex assembly 126

1349

GPI-anchor transamidase complex, GPI8 subunit

GPI-anchor transamidase complex

Distantly related proteases in some bacteria (no COG)

1943

Beta-tubulin folding cofactor D, involved in chromosome segregation

None

2015

NEDD8-activating complex, UBA3 subunit

NEDD8-activating complex

Most bacteria and some archaea (COG0476)

2126

Phosphoethanolamine N-methyltransferase involved in GPI-anchor biosynthesis

Several bacteria and archaea (COG1524)

2884

26S proteasome regulatory complex, subunit RPN10/PSMD4

26S proteasome regulatory complex

No orthologs although von Willebrand A domains are present in a variety of prokaryotic proteins

Contains von Willebrand A domain

2908

26S proteasome regulatory complex, subunit RPN9/PSMD13

26S proteasome regulatory complex

None

Contains PINT domain

0209

Endoplasmic reticulum membrane P-type ATPase

Many bacteria and some archaea (COG0474)

3379

Uncharacterized member of the histidine triad superfamily of nucleotide hydorlases

Most archaea and bacteria (COG0537)

Only biochemical function predicted.

2635

Coatomer (COPI) complex delta subunit

COPI complex

None

2927

Membrane component of ER protein translocation apparatus (Sec62)

Sec complex

None

2978

Dolichol-phosphate mannosyltransferase

All archaea, most bacteria (COG0463)

3198

Signal recognition particle, subunit Srp19

Signal recognition particle

All archaea, no bacteria (COG1400)

3315

Subunit of the targeting complex (TRAPP) involved in ER to Golgi trafficking

TRAPP

None

3369

Subunit of the targeting complex (TRAPP) involved in ER to Golgi trafficking

TRAPP

None

1992

Nuclear export receptor CSE1/CAS (importin beta)

None

New functional predictions

2316

PP-loop family ATP pyrophosphatase domain, which in fungi, plants and insects is fused to a duplicated translation inhibitor domain. The fusion, along with the phyletic pattern of the PP-ATPase domain, suggests an essential function in translation regulation

Orthologs of the PP-loop domain are present in all archaea (COG2102) but not in bacteria. Orthologs of the translation inhibitor domain are found in most bacteria and several archaea (COG0251)

PP-loop ATPases have been previously implicated in base thiolation in various RNAs 127 and proteins in this K/COG might have a similar function, which is likely to be conserved in eukaryotes and archaea. However, the fusion with translation inhibitor, which has been reported to have endoribonuclease activity 128 is a eukaryote-specific feature

2523

Predicted RNA-binding protein containing a PUA domain, probable role in RNA modification 129

Putative novel RNA modification complex

Orthologs present in all archaea (COG2016) but not in bacteria

Several of the archaeal orthologs of this protein form fusions with a PP-loop ATPase domain implicated in base thiolation 127. Thus, the proteins of this KOG might interact with those of KOG2840 (pan-eukaryotic, duplications in Arabidopsis and worm) or KOG2594 (missing in humans and microsporidia) to form a novel enzymatic complex involved in RNA modification

0270, 0271, 1539

WD40-repeat proteins

Processosome

all 0

X,1,X

By analogy with other conserved WD40-repeat proteins, predicted to be subunits of rRNA processing/ribosome assembly complexes

2321

Nucleolar protein, contains WD40 repeats

rRNA processosome?

Probable subunit of an rRNA-processing complex

1763

Uncharacterized conserved protein containing a CCCH Zn-finger; possible role in RNA processing or splicing

None

CCCH fingers have been shown to bind 3' untranslated regions in various mRNAs 130131

2837

Protein containing a U1-type, RNA-binding C2H2 Zn-finger. Probable role in RNA splicing/processing

Spliceosome?

None

U1-type fingers are essential for the assembly of U1 RNP 132

3073

Predicted RNA-binding protein containing PIN domain and involved in 18S rRNA processing

Pre-40S subunit

Most archaea, no in bacteria (COG1412)

Interacts with Nop14p and is required for 40S subunit biogenesis and 18S rRNA maturation (11694595). The presence of the PIN domain suggests RNA-binding and, possibly, RNAse activity

3154

Uncharacterized protein with potential function in translation or ribosomal biogenesis

Pre-40S subunit?

Most archaea, no bacteria (COG2042)

The general functional prediction stems from the observation that the gene for this protein forms a predicted conserved operon with the gene for ribosomal protein L40E in several archaeal genomes

3214

Small protein containing a Zn-ribbon, possibly RNA-binding; potential role in RNA processing or transcription regulation

Conserved in Crenarchaeota (COG4888)

3800

Predicted E3 ubiquitin ligase containing RING finger, subunit of transcription/repair factor TFIIH and CDK-activating kinase assembly factor

TFIIH

None

3176

Predicted α-helical protein, possibly involved in replication/repair; paralog of KOG3636

A novel complex with PCNA involved in replication?

Conserved in most (possibly all) archaea but not in bacteria (COG1711)

A function in DNA replication/repair and/or transcription is suggested by the analysis of the genome context of archaeal orthologs which form an evolutionarily conserved association with the genes for replication sliding clamp (PCNA ortholog) (K.S.M. and E.V.K., unpublished work)

3303

Predicted α-helical protein, possibly involved in replication/repair transcription; paralog of KOG3508

A novel complex with PCNA involved in replication?

Conserved in most (possibly all) archaea but not in bacteria (COG1711)

0396

Predicted E3 ubiquitin ligase

Ub ligase

None

The proteins in this KOG contain a modified RING domain, which might not be capable of metal-binding similarly to the U-box domain 133 that has been shown to function as E3 134

1443

Multitransmembrane protein, predicted drug/metabolite transporter

Most archaea and bacteria (COG0697)

2647

Multitransmembrane protein, potential transporter

Most bacteria and some archaea (COG0628)

2488

Predicted N-acetyltransferase

Most archaea and bacteria (COG0454)

Putative role in ribosomal maturation?

3347

Predicted nucleotide kinase; nuclear protein (Fap7p)

Conserved in all archaea but not in bacteria (COG1936)

Involved in oxidative stress reponse in yeast 135

3974

Predicted sugar kinase

Putative novel complex with KOG2585 proteins

All archaea and most bacteria (COG0063)

Based on fusions seen in prokaryotes, predicted to interact functionally and, possibly, physically with uncharacterized proteins of KOG2585 (represented in all eukaryotes but includes paralogs in some species)

No functional prediction

2318

Uncharacterized conserved protein

None

3237

Uncharacterized conserved protein containing coiled-coil domain

None

Coiled-coil domains are often involved in complex assembly; this could be an uncharacterized component of the chromatin or the spliceosome

*Abbreviations for the functional categories are as in Figure 3. ^†0, essential gene (lethal knockout); 1, non-essential gene (non-lethal knockout); X indicates that no data is available for the given gene. ^‡Data from 85. ^§Data from 86.

Common phyletic patterns of genes that otherwise were not suspected to be functionally linked might suggest the existence of such connections and prompt additional analysis leading to concrete functional predictions 42596061. The pair of KOG5324 and KOG4246 is a case in point that has not been described previously. The initial observation that these KOGs share the same unusual pattern of presence-absence in eukaryotes, and have similar phyletic patterns in prokaryotes, with a ubiquitous presence in archaea, prompted a more detailed examination of the multiple alignments of the respective proteins and the conservation of the (predicted) operon organization in archaea and bacteria (Table 2 and data not shown). The combination of clues from these analyses suggests that the two proteins interact in a still uncharacterized pathway of RNA processing, which also includes RNA 3'-phosphate cyclase (KOG3980)) 62 and cytosine-C5-methylase (NOL1/NOP2 in eukaryotes; KOG1122). The proteins in KOG3833 and KOG4528 are likely to represent novel enzyme families, possibly a kinase-phosphatase pair (E.V.K. and L. Aravind, unpublished data). Notably, these predicted new enzymes are present in animals and E. cuniculi but not in Arabidopsis or yeasts. In contrast, KOG3980 is present in all analyzed eukaryotic genomes except for Arabidopsis, whereas KOG1122 is pan-eukaryotic. These differences in the phyletic patterns of the components of the predicted pathway are concordant with the patterns in eukaryotes in that.

Figure 2 shows the distribution of known and predicted functions of eukaryotic proteins among 20 functional categories for the entire set of KOGs and, separately, for KOGs represented in six or seven species and the animal-specific KOGs. Compared to the functional breakdown of prokaryotic COGs 25, the prevalence of signal transduction is notable among eukaryotes. This feature is particularly prominent in animal-specific KOGs, whereas the highly conserved set is comparatively enriched in proteins that are involved in translation, transcription, chaperone-like functions, cell cycle control and chromatin dynamics (Figure 2). The large number of KOGs for which only general functional prediction was feasible, and those whose functions remain unknown, even among the subset that is represented in six or seven eukaryotic species, emphasizes that our current understanding of eukaryotic biology is seriously lacking with even in respect of the functions of highly conserved genes.

Figure 2

Distribution of the KOGs by the number of paralogs in each of the analyzed eukaryotic genomes

Distribution of the KOGs by the number of paralogs in each of the analyzed eukaryotic genomes. The species abbreviations are as in Figure 1.

The distribution of KOGs by the number of paralogs in each genome is shown in Figure 3. The preponderance of lineage-specific duplication of conserved genes, that is, intra-KOG LSEs, in multicellular eukaryotes is obvious. Cases when a single gene in yeast or, particularly, Encephalitozoon, has two or more co-orthologs in animals and/or plants are most common in KOGs, whereas the reverse situation is rare. These observations support the notion of the major contribution of LSE to the evolution of eukaryotic complexity 52. However, 131 KOGs are represented by a single ortholog in all genomes compared (Table 2) and a substantial number of KOGs have one member from a majority of the genomes (data not shown). Recent theoretical modeling of the evolution of paralogous families has suggested that, in general, ancient protein families tend to have multiple paralogs 563. Therefore, whenever a KOG has a single member in all or most species, this should be attributed to selection against duplication of this particular gene. A prominent cause of such selection could be the involvement of the respective gene products in essential multisubunit complexes, such that imbalance between subunits leads to deleterious effects 64.

Figure 3

Functional breakdown of the KOGs

Functional breakdown of the KOGs. Designations of functional categories: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell-cycle control and mitosis; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism; I, lipid metabolism; J, translation; K, transcription; L, replication and repair; M, membrane and cell wall structure and biogenesis; O, post-translational modification, protein turnover, chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; T, signal transduction; U, intracellular trafficking and secretion; Y, nuclear structure; Z, cytoskeleton; R, general functional prediction only (typically, prediction of biochemical activity), S, function unknown. This breakdown is only for KOGs that included at least three species.

Known and new functions of single-member, pan-eukaryotic KOGs

We examined in greater detail the 131 KOGs that are represented by a single gene in each of the seven genomes (Table 2). As can be envisaged from their presence in diverse eukaryotic taxa, including the 'minimal' genome of Encephalitozoon, and as shown by comparison with the knockout phenotype data (Table 2 and see below), these pan-eukaryotic KOGs are of particular biological importance. For the great majority of these KOGs (113 of the 131), the function has been experimentally determined or confidently predicted to a varying degree of detail using computational methods (Table 2). However, around 20 KOGs from this set remained uncharacterized at the time of this analysis and, for all but two of these, substantial functional inferences could be drawn through a combination of sequence-profile analysis, structure prediction and genomic-context analysis of prokaryotic homologs (Table 2). Some of these predicted new functions are variations on well-known themes, such as two predicted PP-loop ATPases, which are probably involved in novel, essential RNA modifications (KOGs 2522 and 2316) or two predicted E3 components of ubiquitin ligases (KOGs 0396 and 3800). Other predicted functions appear to be completely new, such as proteins in KOG3176 and 3303 which are likely to be essential components of eukaryotic replication and/or repair systems. Each of these uncharacterized but ubiquitous and largely essential eukaryotic genes is an attractive target for experimental studies.

Examination of the experimentally characterized and predicted functions of pan-eukaryotic, single-member KOGs leads to interesting conclusions. Nearly all the functionally characterized KOGs in this set consist of proteins that are subunits of known multiprotein complexes (Table 2). The most prominent of these are the complexes involved in rRNA processing and ribosome assembly, such as the recently discovered rRNA processosome and the pre-40S subunit, as well as the spliceosome, and various complexes involved in transcription (Table 2). Accordingly, this set of KOGs is markedly enriched for proteins involved in various forms of RNA processing, assembly of ribonucleoprotein (RNP) particles and transcription. In addition, KOGs in the single-member pan-eukaryotic set include subunits of molecular complexes that are not directly related to RNA processing, such as the proteasome, the TCP-1 chaperonin complex 65 and the TRAPP complex involved in protein trafficking 66. Altogether, more than 80% of the yeast proteins in the pan-eukaryotic, single-member KOGs belong to known macromolecular complexes included in the MIPS database 67, as compared to around 64% for all yeast proteins in the KOGs, which is a moderate but statistically highly significant excess (data not shown). This preponderance of multiprotein complex formation among the single-member pan-eukaryotic KOGs is fully compatible with the balance hypothesis 64.

The most unexpected observation regarding the single-member, pan-eukaryotic KOGs, is probably that in 14 of these proteins, the only detectable domain was the WD40 repeat (Table 2). This is particularly notable because WD40-repeat proteins, which are extremely abundant in eukaryotes and are present in several prokaryotic lineages as well 68, are not generally known to form well-defined, one-to-one orthologous relationships. The WD40 proteins in the pan-eukaryotic KOGs listed in Table 2 are exceptions, which is probably due to their unique and essential roles in the assembly of RNA-processing complexes. It has recently been demonstrated that, in S. cerevisiae, seven of these proteins are subunits of the 18S rRNA processosome, or at least are involved in ribosomal assembly 6970. Taking these results together with the unusual phyletic pattern, it seems possible to predict with considerable confidence that those WD40 proteins in the 131-KOG set that remain uncharacterized belong to the same or similar RNA-processing complexes (Table 2).

With some notable exceptions, such as the WD40 proteins, the KOGs in the single-member, pan-eukaryotic set show remarkable patterns of evolutionary conservation: they are either (nearly) ubiquitous in the three kingdoms of life, for example, RNA polymerase subunits, or are universally conserved in eukaryotes and archaea but missing in bacteria, such as most of the proteins implicated in RNA processing (Table 2). Thus, it appears that elaborate molecular machines central to the functioning of the eukaryotic cell have evolved, largely from ancestral archaeo-eukaryotic components, at the onset of eukaryotic evolution, and both loss and duplication of the respective genes have been strongly selected against throughout the rest of eukaryotic evolution.

Variation of evolutionary rates among KOGs

Genome-wide analysis of protein evolutionary rates shows a broad range of variation 71. Here, we investigate the variation of evolutionary rates among the ubiquitous KOGs represented in all seven analyzed genomes and the connection between the evolutionary rate and protein function in the KOG set. The characteristic evolutionary rate of each KOG, which included a member(s) from Arabidopsis, was determined by measuring the mean evolutionary distance from Arabidopsis (the outgroup in the phylogenetic tree; see below) to the other species. Even among the KOGs that include all seven species and, accordingly, appear to represent the conserved core of eukaryotic genes, the evolutionary rates differ by a factor of 20 between the fastest- and the slowest-evolving KOGs. Excluding 5% of the KOGs from each tail of the distribution still leaves almost a fourfold difference in evolutionary rates (Figure 4a).

Figure 4

Variation of amino-acid substitution rates among KOGs

Variation of amino-acid substitution rates among KOGs. (a) Probability-density function for the distribution of evolutionary rates among the set of KOGs including all seven analyzed eukaryotic species. (b) Distribution functions for the evolutionary rates in different functional categories of KOGs. The designations of functional categories are as in Figure 3.

We then compared the distributions of evolutionary rates for different functional categories of KOGs (Tables 3,4 and Figure 4b). Although all the distributions substantially overlapped, there was a statistically highly significant difference between the evolutionary rates for proteins with different functions (Tables 3,4 and Figure 4b). The slowest-evolving proteins are those involved in translation and RNA processing, the fastest-evolving ones are involved in cellular trafficking and transport, whereas components of replication and transcription systems have intermediate evolutionary rates (Tables 3,4 and Figure 4b).

Table 3

Evolutionary rates in KOGs with different functions: evolutionary rates for different functional categories of KOGs*

Functional category

Number of KOGs

Mean rate, substitutions per site

Standard deviation

227

0.98

0.37

0.98

0.30

167

1.01

0.36

140

1.01

0.43

307

1.01

0.40

1.05

0.34

130

1.07

0.38

139

1.11

0.38

1.13

0.33

1.13

0.46

209

1.15

0.42

115

1.16

0.43

110

1.16

0.32

200

1.18

0.39

111

1.19

0.40

415

1.23

0.42

1.26

0.47

196

1.27

0.42

1.27

0.37