Evolution of selenophosphate synthetases: emergence and relocation …public-files.prbb.org/publicacions/55dc77e0-135b-0133-59... · 2016. 8. 4. · 111 human SPS1 interacts with

1

Evolution of selenophosphate synthetases: emergence and relocation 1

of function through independent duplications and recurrent 2

subfunctionalization 3

Marco Mariotti1, 2, 3, 4*

, Didac Santesmasses1, 2, 3

, Salvador Capella-Gutierrez1, 2

, Andrea 4

Mateo5, Carme Arnan

1, 2, 3, Rory Johnson

1, 2, 3, Salvatore D’Aniello

6, Sun Hee Yim

4, 5

Vadim N Gladyshev4, Florenci Serras

5, Montserrat Corominas

5, Toni Gabaldón

1, 2, 7, 6

Roderic Guigó1, 2, 3*

7

*. Corresponding author; email: [email protected] or [email protected] 8

1. Bioinformatics and Genomics Programme, Centre for Genomic Regulation 9

(CRG), Dr. Aiguader 88, 08003 Barcelona, Catalonia, Spain. 10

2. Universitat Pompeu Fabra (UPF), 08003 Barcelona, Catalonia, Spain. 11

3. Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), 08003 Barcelona, 12

Catalonia, Spain 13

4. Division of Genetics, Department of Medicine, Brigham & Women’s Hospital, 14

Harvard Medical School, Boston, MA 02115, USA 15

5. Departament de Genètica, Facultat de Biologia and Institut de Biomedicina 16

(IBUB) de la Universitat de Barcelona (UB), 08028 Barcelona, Catalonia, Spain 17

6. Department of Biology and Evolution of Marine Organisms, Stazione Zoologica 18

Anton Dorhn, Villa Comunale, 80121, Napoli, Italy. 19

7. Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluís 20

Companys 23, 08010 Barcelona, Catalonia, Spain. 21

22

Running Title: Phylogeny of selenophosphate synthetases 23

Keywords: selenocysteine, gene duplication, subfunctionalization, 24

evolution of function, readthrough. 25

Abstract 26

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Selenoproteins are proteins that incorporate selenocysteine (Sec), a non-standard 28

amino acid that is encoded by UGA, normally a stop codon. The synthesis of Sec 29

requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all 30

prokaryotic and eukaryotic genomes encoding selenoproteins. Here we study the 31

evolutionary history of SPS genes, providing a map of selenoprotein function spanning 32

the whole tree of life. SPS is itself a selenoprotein in many species, though functionally 33

equivalent homologues that replace the Sec site with cysteine (Cys) are common. Many 34

metazoans, however, possess SPS genes with substitutions other than Sec or Cys 35

(collectively referred to as SPS1). Using complementation assays in fly mutants, we 36

show that these genes share a common function, which appears to be distinct from the 37

synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed 38

SPS2), and unrelated to Sec synthesis. Even though sharing the same function, we 39

show here that SPS1 genes originated through a number of independent gene 40

duplications from an ancestral metazoan selenoprotein SPS2 gene, that most likely 41

already carried the SPS1 function. Thus, in SPS genes, parallel duplications and 42

subsequent convergent subfunctionalization have resulted in the segregation to different 43

loci of functions initially carried by a single gene. A key actor in the evolution of SPS 44

genes is a stem-loop RNA structure enhancing the readthrough of the Sec-UGA codon, 45

whose origin may be traced back to prokaryotes. The evolutionary history of SPS 46

constitutes a remarkable example of emergence and evolution of gene function, which 47

we have been able to trace with unusual detail thanks to the singular features of SPS 48

genes, wherein the amino acid at a single site determines unequivocally protein function 49

and is intertwined to the evolutionary fate of the entire selenoproteome. 50




3

Introduction 51

Selenoproteins are proteins that incorporate the non-standard amino acid 52

selenocysteine (Sec or U) in response to the UGA codon. The recoding of UGA, 53

normally a stop codon, to code for Sec is arguably the most outstanding programmed 54

exception to the genetic code. Selenoproteins are found, albeit in small numbers, in 55

organisms across the entire tree of life. Recoding of UGA is mediated by RNA structures 56

within selenoprotein transcripts, the SECIS (SElenoCysteine Insertion Sequence) 57

elements. Sec biosynthesis and insertion also require a dedicated system of trans-acting 58

factors that include elements that are common and others that are specific to the three 59

domains of life: bacteria (Kryukov and Gladyshev 2004)(Yoshizawa and Bock 2009), 60

archaea (Rother et al. 2001), and eukaryotes (Squires and Berry 2008)(Allmang et al. 61

2009). 62

The very existence of selenoproteins is puzzling. Sec can apparently be substituted by 63

cysteine (Cys)—as often happens during evolution (Zhang et al. 2006)(Chapple and 64

Guigó 2008)(Mariotti et al. 2012)— with seemingly a small or null impact on protein 65

function. In fact, selenoproteins may be absent in an entire taxonomic group, but present 66

in sister lineages. This can be seen most dramatically within fruit flies: while Drosophila 67

melanogaster and most other flies possess three selenoprotein genes, their relative 68

Drosophila willistoni has replaced Sec with Cys in them, and lost the capacity to 69

synthesize Sec (Chapple and Guigó 2008)(Lobanov et al. 2008). Fungi and plants have 70

also lost this capacity (Lobanov et al. 2009). In other cases, however, such as in 71

Caenorhabditis elegans, the entire pathway is maintained only to synthesize a single 72

selenoprotein (Taskov et al. 2005). It appears that selective pressure exists to maintain 73

Sec, at least in vertebrates, since strong purifying selection across Sec sites that prevent 74

mutations to Cys has been reported (Castellano et al. 2009). Sec encoding has been 75

hypothesized to be an ancestral trait, already present in the early genetic code, since a 76

number of selenoprotein families are shared between prokaryotes and eukaryotes. 77

However, the evolutionary continuity of the Sec recoding systems across domains of life 78

is not certain, since it would require the translocation of the SECIS element (within the 79

coding region in bacteria but within the 3’ UTR in eukaryotes), as well as the radical 80

alteration of its structure. 81

Selenophosphate synthetase (SPS, also called SelD or selenide water dikinase) is 82




4

unique among the components of the Sec biosynthesis machinery in that it is often a 83

selenoprotein itself. SPS catalyzes the synthesis of selenophosphate from selenide, ATP 84

and water, producing AMP and inorganic phosphate as products. Selenophosphate is 85

the selenium donor for the synthesis of Sec, which, in contrast to other amino acids, 86

takes place on its own tRNA, tRNAsec (Xu et al. 2007a)(Palioura et al. 2009). 87

SPS proteins are conserved from bacteria to human with about 30% identity and are 88

found in all species known to encode selenoproteins. In prokaryotes, SPS (i.e., SelD) is 89

found also in species where selenophosphate is used to produce selenouridine in tRNAs 90

(SeU). In these species, it acts as the selenium donor to protein ybbB (selenouridine 91

synthase). The presence of the two traits (SeU and Sec) overlaps, but not completely 92

(Romero et al. 2005). In eukaryotes, SPS is generally found as a selenoprotein, while in 93

prokaryotes homologues with Cys aligned to the Sec position are common. As for all 94

selenoproteins, Sec and Cys homologues of SPS are expected to perform the same 95

molecular function, although catalytic efficiency can vary. Indeed, selenophosphate 96

synthesis activity has been demonstrated experimentally for various Sec- and Cys- SPS 97

proteins, as well as for artificial Cys mutants (Kim et al. 1997)(Persson et al. 1997)(Xu et 98

al. 2007a). 99

In vertebrates and insects, two paralogous SPS genes have been reported: SPS2 (i.e., 100

Sephs2), which is a selenoprotein, and SPS1 (i.e., Sephs1), which is not, and carries a 101

threonine (Thr) in vertebrates and an arginine (Arg) in insects in place of Sec (Xu et al. 102

2007b). In contrast to Cys conversion, Thr or Arg conversion in SPS1 seems to result in 103

the abolishment of the selenophosphate synthase function. Indeed, murine SPS1 does 104

not generate selenophosphate in vitro, and does not even consume ATP in a selenium 105

dependent manner (Xu et al. 2007a). Consistently, selenoprotein synthesis is unaffected 106

in a knockout of SPS1 in mouse cell lines (Xu et al. 2007b). Similarly, Drosophila SPS1 107

(i.e., ptuf/SelD) lacks the ability to catalyze selenide-dependent ATP hydrolysis or to 108

complement SelD deficiency in Escherichia coli (Persson et al. 1997). In insects, SPS1 109

is preserved in species that lost selenoproteins (Chapple and Guigó 2008). Although 110

human SPS1 interacts with Sec synthase (SecS) (Small-Howard et al. 2006), these 111

findings, taken as a whole, suggest that SPS1 functions in a pathway unrelated to 112

selenoprotein biosynthesis (Lobanov et al. 2008). What the function of SPS1 may be 113

remains an open question. Human SPS1 has been proposed to function in Sec 114

recycling, since a E. coli SelD mutant can be rescued by SPS1 but only when grown in 115




5

the presence of L-selenocysteine (Tamura et al. 2004). In Drosophila, SPS1 has been 116

proposed to be involved in vitamin B6 metabolism (Lee et al. 2011) and in redox 117

homeostasis, since it protects from damage induced by reactive oxygen species (ROS) 118

(Morey et al. 2003). 119

Here we study the evolutionary history of SPS genes across the tree of life. We found 120

that the presence of Sec/Cys SPS genes, together with a few other gene markers, 121

recapitulates well the selenium utilization traits (Sec and SeU) in prokaryotic genomes. 122

Within eukaryotes, specifically within metazoans, we detected a number of SPS 123

homologues with amino acids other than Sec or Cys at the homologous UGA position. 124

We found that Cys- or Sec-containing SPS genes (SPS2) are found in all genomes 125

encoding selenoproteins, while genomes that contain only SPS genes carrying amino 126

acids other than Sec or Cys at the homologous UGA position (SPS1) do not encode 127

selenoproteins. In SPS proteins, thus, it appears that the residue occurring at a single 128

site is a precise marker of function, which can be easily traced in genomes by searching 129

for selenoprotein genes and other markers of selenium utilization. This feature, that may 130

be unique among all protein families, makes SPS genes singularly appropriate to 131

investigate the evolution of gene function. Here, thanks to this feature, we have been 132

able to untangle the complex history of SPS genes with great detail. Our analysis 133

reveals that SPS1 genes in different metazoan lineages (including those of human and 134

fly) originated via parallel duplications from an ancestral Sec-carrying SPS gene. Despite 135

their independent origin, SPS1 genes share similar evolutionary constraints, and have a 136

common function, likely present in the ancestral metazoan SPS2 gene. This indicates 137

selective pressure during metazoan history to segregate different functions to separate 138

loci, and constitutes a remarkable example of recurrent escape from adaptive conflict 139

through gene duplication and subfunctionalization (Hittinger and Carroll, 2007). Within 140

insects, the SPS duplication was followed by the loss of the Sec encoding SPS2 gene in 141

several lineages, which therefore lost the capacity to synthesize selenoproteins 142

(Chapple and Guigó 2008)(Lobanov et al. 2008)—becoming, together with some 143

nematodes (Otero et al. 2014), the only known selenoproteinless metazoans. Strikingly, 144

SPS1 conserved the ancestral UGA codon in selenoproteinless Hymenoptera. Our 145

analyses point out that UGA readthrough in hymenopterans is enhanced by overlapping 146

RNA structures, also present in other selenoproteins. These structures could be related 147

to bacterial SECIS elements, uncovering a possible evolutionary link between the 148

prokaryotic and eukaryotic Sec encoding systems. They would have played a key role 149




6

throughout all the evolution of SPS genes, and particularly in the emergence of the 150

SPS1 function in the metazoan ancestral Sec-carrying SPS gene. 151

Results 152

Selenoprotein genes are usually miss-annotated in prokaryotic and eukaryotic genomes, 153

due to the recoding of UGA, normally a stop codon, to Sec. Therefore, we used 154

Selenoprofiles (Mariotti and Guigó 2010), a computational tool dedicated to the 155

prediction of selenoproteins and selenoprotein homologues. We have run Selenoprofiles 156

to search for SelD/SPS genes in all available fully-sequenced eukaryotic and prokaryotic 157

genomes, 505 and 8,263, respectively. We then utilized a combination of approaches to 158

reconstruct their phylogenetic history. Methods and analyses are fully discussed in 159

Supplementary Material S1-S6. 160

SelD as a marker for selenium utilization in prokaryotes 161

Figure 1 shows the distribution of SelD (i.e., prokaryotic SPS) genes in a reference set of 162

223 prokaryotic genomes (Pruitt et al. 2012), along with the presence of other selenium 163

utilization gene markers such as the bacterial selenocysteine synthase gene (SelA). The 164

occurrence of SelD and other Sec machinery components is in good agreement with 165

previous findings (Zhang et al. 2006)(Zhang et al. 2008)(Zhang and Gladyshev 166

2008)(Zhang et al. 2010). Supplementary Material S1 contains details of the genes 167

found in each major lineage investigated, both in the reference set, and in the extended 168

set of 8,263 genomes. 169

SelD genes were found in 26% of the reference prokaryotic genomes. A considerable 170

fraction (19%) of the detected SelD genes encoded a protein with a Sec residue (always 171

in the same position), with all the rest containing Cys instead. The occurrence of SelD is, 172

in general, consistent with the other gene markers for selenium utilization and also with 173

selenoprotein presence (see Figure 1), with only a few exceptions (Supplementary 174

Material S1). The Sec trait (SelD, SelA, tRNAsec, selenoproteins) was found in a slightly 175

larger group of organisms than the SeU trait (SelD, ybbB): 18% vs 16%, respectively. 176

The two traits showed a highly significant overlap: 10% of all species had both (p-value < 177

0.0001, one-tailed Fisher’s exact test). The Sec and SeU markers showed a scattered 178

distribution across the prokaryotic tree, reflecting the dynamic evolution of selenium 179

utilization. The complexity of this phylogenetic pattern is even more evident when 180

considering the extended set of prokaryotic species (see Supplementary Material S1 and 181




7

Supplementary Figure SM1.1). 182

In almost every species with SelD (93%), genes for SelA and/or ybbB were identified, 183

supporting the utilization of selenophosphate for Sec and SeU. A notable exception was 184

the Enterococcus genus, where many species including Enterococcus faecalis 185

possessed SelD, but no other markers of selenium usage. This had already been 186

reported as an indicator of a potential third selenium utilization trait (Romero et al. 187

2005)(Zhang et al. 2008). Selenium is in fact used by these species as a cofactor to 188

molybdenum hydroxylases (Haft and Self 2008)(Srivastava et al. 2011). 189

In Pasteurellales, an Order within Gammaproteobacteria, we identified a bona-fide Cys 190

to Sec conversion. Most of Gammaproteobacteria possess a SelD-Cys (or none), and 191

Sec forms are found almost uniquely in Pasteurellales. Phylogenetic sequence signal 192

supports codon conversion rather than horizontal transfer as the cause for SelD-Sec 193

(Supplementary Material S1). Even though cases of conversion of Cys to Sec have been 194

proposed (Zhang et al. 2006), this is the first clearly documented case. Among archaea, 195

SelD was found only in Methanococcales and Methanopyri genomes, whose 196

selenoproteins have been previously characterized (Stock and Rother 2009). The SeU 197

trait was found only in Methanococcales, although with a peculiarity: ybbB is split in two 198

adjacent genes (Su et al. 2012). 199

Previous reports have described a number of SPS genes fused to other genes (Zhang et 200

al. 2008)(da Silva et al. 2013). Thus, we used a computational strategy to identify SelD 201

gene fusions or extensions (see Methods and Supplementary Material S2). Fusions with 202

a NADH dehydrogenase–like domain (Zhang et al. 2008) are by far the most common, 203

and they are found scattered across a wide range of bacteria (Supplementary Figure 204

SM2.1). We also detected two instances of fusions with the NifS-like protein (Cys 205

sulfinate desulfinase, proteins that deliver selenium for the synthesis of selenophosphate 206

by SPS2, (Lacourciere et al. 2000)). In all cases, the extension/fusion is on the N-207

terminal side of the SPS genes, and these are always SelD-Cys with the single 208

exception of NifS-SPS in Geobacter sp. FRC-32, which contains Sec. Since we found 209

selenoproteins and other Sec machinery genes in all these genomes, we predict that 210

generally these extended SPS genes have retained the original selenophosphate 211

biosynthetic activity. 212

SPS2 as a marker for Sec utilization in eukaryotes 213




8

Figure 2 shows SPS genes and predicted selenoproteins found in a representative set of 214

eukaryotic genomes. The presence of SPS2 genes (defined as those with Sec, or Cys) 215

correlates perfectly with the presence of selenoproteins. Thus, SPS2 is a marker of Sec 216

utilization in eukaryotes. Our results replicate and substantially expand previous surveys 217

of selenoproteins in eukaryotic genomes (e.g. (Lobanov et al. 2007)(Chapple and Guigó 218

2008)(Lobanov et al. 2009)(Jiang et al. 2012)). 219

Overall, the Sec trait exhibits a rather scattered distribution in protists reflecting a 220

dynamic evolution similar to bacteria. We found SPS2 (and therefore selenoproteins) 221

scattered across Stramenopiles, Alveolata, Amoebozoa and other protist lineages, 222

presumably due to multiple independent events of selenoprotein extinction. In contrast, 223

we found selenoproteins in all investigated Kinetoplastida (Euglenozoa), including the 224

parasites Trypanosoma and Leishmania (Lobanov et al. 2006)(Cassago et al. 2006). We 225

did not find any bona fide SPS2 nor selenoproteins in Fungi or land plants 226

(Embryophyta), despite many genomes were searched (284 and 41, respectively, a 227

subset of which are shown in Figure 2). In contrast, green algae genomes contain large 228

numbers of selenoproteins as previously reported (Novoselov et al. 2002)(Palenik et al. 229

2007). The largest number was in the pelagophyte algae Aureococcus anophagefferens 230

(heterokont, Stramenopiles), known for its rich selenoproteome (Gobler et al. 2013). All 231

metazoans encode SPS2 and selenoproteins, with exceptions detected so far only in 232

some insects (Chapple and Guigó 2008)(Lobanov et al. 2008) and some nematodes 233

(Otero et al. 2014). 234

As in prokaryotes, we found a few protist genomes in which SPS2 is fused to other 235

genes. Fusions with a NADH dehydrogenase–like domain are also the most common, 236

and scattered among many taxa (Figure 2). We detected NifS-SPS fusions in the 237

amoeba Acanthamoeba castellani and the heterolobosean Naegleria gruberi. In these 238

two genomes, we found additional SPS2 candidates (Supplementary Material S2). In N. 239

gruberi, SPS2 is fused to a polypeptide containing a methyltransferase domain (da Silva 240

et al. 2013). Finally, all SPS2 proteins in Plasmodium species were found to possess a 241

large polypeptide extension (>500 amino acids). This domain shows no homology with 242

any known protein, and its function remains unknown. We did not find convincing SPS 243

fusions in non-protist eukaryotes (see Supplementary Material S2). 244

The reconstructed gene tree of the bacterial, archaeal and eukaryotic SPS sequences 245

follows broadly their known phylogenetic relationships (Supplementary Figure SM3.1), 246




9

and supports the continuity of the selenoprotein system across the three domains of life. 247

Most likely, thus, the last common ancestor of eukaryotes and prokaryotes possessed 248

selenoproteins and SPS, probably as a selenoprotein itself. The continuity in SPS 249

phylogenetic signal is apparently broken only in few protist lineages, which seem to have 250

acquired a bacterial-like SPS gene by horizontal transfer (see Supplementary Material 251

S3). All the gene fusions shared by protists and bacteria (NADH-SPS, NifS-SPS) are 252

explained by this process (i.e., they are not independently evolved fusions but the result 253

of a fusion event in bacteria which was subsequently transmitted horizontally). 254

Independent duplications of SPS2 generates SPS1 proteins in 255

metazoans 256

In many metazoan lineages we detected additional SPS genes, which are neither 257

selenoproteins nor Cys-homologues. Since, within metazoans, SPS-Cys are found only 258

in nematodes, and, outside metazoans, additional SPS genes are absent (Figure 2), we 259

argue that the last common metazoan ancestor possessed a single SPS gene with Sec 260

(i.e., it was SPS2). Our results show that the additional SPS genes do not have a single 261

origin, but instead they were generated by independent duplications of SPS2 in a 262

number of metazoan lineages (Supplementary Material S3). We have specifically 263

identified four independent duplications (Figures 3 and 4). One SPS duplication occurred 264

at the root of the vertebrates, probably as part of one of the reported rounds of whole 265

genome duplications (Dehal and Boore, 2005). Another duplication occurred within 266

tunicates, likely originated by retro-transposition of an alternative isoform of SPS2. 267

Another duplication occurred within annelids, at the root of the Clitellata lineage. Finally, 268

a duplication occurred at the root of insects. In each of these duplications, a specific 269

substitution of the Sec residue was fixed: threonine in vertebrates, glycine in tunicates, 270

and leucine in annelids. In insects, however, the UGA codon was maintained after 271

duplication, and substituted in some lineages by arginine. There were at least two 272

independent UGA to arginine substitutions in Paraneoptera and Endopterygota. 273

Remarkably, insects without selenoproteins lost the original SPS2 gene, but maintained 274

the duplicated copy. 275

Therefore, as a universal trend, Cys- or Sec- containing SPS genes (to which we refer 276

as SPS2) are found in all metazoan (eukaryotic) genomes encoding selenoproteins, 277

while eukaryotic genomes containing only SPS genes carrying amino acids other than 278

Sec or Cys at the homologous UGA position do not encode selenoproteins. Thus, the 279




10

duplicated non-Cys, non-Sec copies of SPS2 in human and fly are unlikely to carry a 280

function related to selenoprotein synthesis. Since in human and fly, they are commonly 281

referred to as SPS1 (Xu et al. 2007b), we will collectively refer to all non-Cys, non-Sec 282

SPS2 duplications in metazoans as SPS1 (e.g. SPS1-Thr for human SPS1). 283

In the next section, we briefly describe each SPS duplication separately. 284

SPS phylogeny in vertebrates 285

All non-vertebrate deuterostomes (except tunicates) and Cyclostomata (jawless 286

vertebrates, such as lampreys) encode only one SPS2 gene, while all Gnasthostomata 287

possess SPS1 in addition. Supported also by a strong phylogenetic signal 288

(Supplementary Figure SM3.2), we conclude that vertebrate SPS1 (SPS1-Thr) 289

originated from a duplication of SPS2 concomitant with conversion of Sec to threonine at 290

the root of Gnasthostomata (Supplementary Material S3). The conservation of intron 291

positions within the protein sequence is consistent with the duplication involving the 292

whole gene structure, and given its phylogenetic position, this may have occurred 293

through one of the reported rounds of whole genome duplications at the base of 294

vertebrates (Dehal and Boore, 2005). As recently reported (Mariotti et al. 2012), in 295

mammals the SPS2 gene duplicated again, this time by retro-transposition, generating a 296

second SPS2-Sec gene almost identical to the parental, except for the lack of introns. In 297

placental mammals, the intronless SPS2 replaced functionally the parental gene (which 298

was lost), while non-placental mammals still retain the two copies (see, for example, 299

Monodelphis domestica in Figure 2). 300

SPS phylogeny in tunicates 301

Tunicates are the closest outgroup to vertebrates (Delsuc et al. 2006), with ascidians 302

(sea squirts) constituting the best-studied and most sequenced lineage. In the ascidian 303

Ciona, we identified a single SPS gene. This appears to be the direct descendant of the 304

ancestral metazoan SPS2, and possesses a SECIS element in the 3’UTR (necessary for 305

the incorporation of Sec). Nonetheless, this gene produces two different protein 306

isoforms, deriving from alternative exon structures at the 5’ end (SPS-ae; Supplementary 307

Material S4). One isoform carries Sec (SPS-Sec, corresponding to the SPS2), while the 308

other one, previously unreported, has a glycine instead (SPS-Gly, corresponding to 309

SPS1). We mapped the origin of the SPS-Gly isoform to the root of ascidians, since the 310

non-ascidian tunicate Oikopleura dioica, appears to have only SPS2-Sec gene with a 311




11

single isoform, while both isoforms (SPS-Sec and SPS-Gly) are found in the ascidian 312

Molgula tectiformis (see Figure 5). We also found both forms in the recently sequenced 313

ascidian species Botryllus schlosseri (Voskoboynik et al. 2013) and Halocynthia roretzi, 314

belonging to the sister lineages of Styelidae and Pyuridae, respectively. However, in 315

these species the two forms mapped to distinct genomic loci, and they correspond 316

therefore to two different genes (see Supplementary Material S4). SPS-Sec is intronless, 317

and contains a SECIS within the 3’UTR. It corresponds, thus, to SPS2. SPS-Gly 318

possesses instead the ancestral intron structure (very similar to O.dioica SPS2), and has 319

no SECIS. It corresponds, therefore, to SPS1. Most likely, the ancestral SPS-sec 320

alternative transcript isoform retro-transposed to the genome at the root of Styelidae and 321

Pyuridae. This generated a copy that soon replaced functionally the SPS-Sec isoform of 322

the parental gene, which as a result specialized in the production only of the SPS-Gly 323

isoform, as both the Sec coding exon and the SECIS element degenerated. This 324

exemplifies an evolutionary scenario, not frequently reported in the literature, in which 325

alternative transcripts precede gene duplication, providing a possible intermediary step 326

of how a dual-function protein can escape from adaptive conflict (Hittinger and Carroll 327

2007). 328

SPS phylogeny in insects 329

Insects provide a unique framework to study selenoprotein evolution. They have 330

undergone several waves of complete selenoprotein extinction, in which selenoprotein 331

genes were converted to Cys homologues or lost, and the Sec machinery degenerated 332

and/or disappeared. This process occurred in several lineages independently: 333

Hymenoptera, Lepidoptera, Coleoptera (or at least Tribolium castaneum), in the 334

Drosophila willistoni lineage (Chapple and Guigó 2008)(Lobanov et al. 2008) within 335

Endopterygota, and also in the paraneopteran pea aphid (Acyrthosiphon pisum (The 336

International Aphid Genomics Consortium 2010)). Consistent with its function, the SPS-337

Sec (SPS2) gene is present in every insect genome coding for selenoproteins, and it is 338

absent in every insect genome not coding for selenoproteins (Figure 2). SPS1 genes, in 339

contrast, are present in all insect genomes. Most insect SPS1 genes (in Lepidoptera, 340

Coleoptera, Diptera, etc) use arginine at the Sec/Cys site (SPS1-Arg, Figures 3 and 4). 341

Instead, SPS1 has a UGA codon at this position in Hymenoptera, and in two non-342

monophyletic species within paraneopterans: Rhodnius prolixus and Pediculus 343

humanus. In these, however, and in contrast to hymenopterans, we found an additional 344




12

SPS-Sec gene with SECIS element (SPS2), as well as, consistently, a number of other 345

selenoproteins and the complete Sec machinery. From all these data (see also 346

Supplementary Material S3), we hypothesize (Figure 3) that all insect SPS1 genes 347

derive from the same SPS2 duplication event occurred approximately at the root of 348

insects, initially generating a UGA-containing, SECIS lacking gene (SPS1-UGA). In most 349

lineages the new gene switched the UGA codon to arginine generating SPS1-Arg 350

proteins. This occurred at least twice independently, in the pea aphid and in the last 351

common ancestor of Coleoptera, Lepidoptera, and Diptera. In Hymenoptera and most 352

Paraneoptera, the gene is still conserved with UGA and no SECIS. The original SPS2 353

was lost in all lineages where selenoproteins disappeared. 354

SPS1-UGA: non-Sec readthrough 355

The strong conservation of the UGA codon in hymenopteran/paraneopteran SPS1-UGA 356

aligned exactly at the position of the SPS2 Sec-UGA codon is extremely puzzling. SPS1-357

UGA does not contain a SECIS element. Furthermore, Hymenoptera lack most 358

constituents of the Sec machinery, and these organisms cannot synthesize 359

selenoproteins. However, the striking conservation of the insect SPS1-UGA sequence 360

strongly indicates that it is translated and functional. Previously, we had hypothesized 361

that SPS1-UGA could perhaps be translated by a readthrough mechanism not involving 362

Sec insertion (Chapple and Guigó 2008). In this respect, there is growing evidence for 363

abundant stop codon readthrough in insects, with UGA being the most frequently 364

observed readthrough codon in Drosophila (Jungreis et al. 2011). 365

Here, we have found additional strong evidence in support of the translational recoding 366

of SPS1-UGA. First, all ten recently sequenced hymenopteran genomes show a clear 367

pattern of protein coding conservation across the UGA, resulting in a readthrough 368

protein of an approximate size of SPS. 369

Second, we found the hexanucleotide GGG-UG[C/U], which is highly overrepresented 370

next to known viral “leaky” UAG stop codons (Harrell et al. 2002), to be ultraconserved 371

subsequent to the UGA in SPS1-UGA genes. While the hexanucleotide is found 372

scattered in some other metazoan SPS2 sequences—where it could actually contribute 373

to UGA translation—it is absent from all insect SPS2 genes (Figure 6). Moreover, the 374

hexanucleotide is also absent from insect SPS1 genes having Arg instead of UGA. 375

Third, SPS1-UGA contains a conserved secondary structure overlapping the UGA (and 376




13

therefore the hexanucleotide). We will name this structure HRE, for hymenopteran 377

readthrough element. For its location and structure (Supplementary Material S6, 378

Supplementary Figure SM6.2), HRE appears to derive from stem-loop structures called 379

SRE (from Sec redefinition elements), previously identified in many selenoprotein genes 380

including SPS2 (Howard et al. 2005), and that promote readthrough activity. Indeed, the 381

SRE element of human Selenoprotein N gene (SepN1) was functionally characterized 382

(Howard et al. 2005)(Howard et al. 2007), showing that it promotes recoding of UGA 383

codons. In the presence of a downstream SECIS element, Sec insertion is enhanced. In 384

its absence, a Sec-independent readthrough activity is still observed. We hypothesize 385

that HRE of SPS1-UGA has a similar activity. In further support of this, we used RNAz 386

(Gruber et al. 2010) to characterize the secondary structures embedded in the coding 387

sequence of all SPS genes (see Methods and Supplementary Material S6). In 388

prokaryotes, this yielded the bacterial SECIS of the Sec containing SelD genes 389

(Supplementary Figure SM6.1). In eukaryotes, we obtained stable stem loops (SRE) in 390

the same region of all UGA-containing SPS genes (Supplementary Figure SM6.2). 391

Strikingly, the largest and most stable structures were in SPS1-UGA, where we 392

predicted HREs as a 3 stem clover-like structure with the UGA in the apex of the middle 393

stem. 394

Overall, these results strongly suggest that the insect SPS1-UGA gene is translated. 395

Furthermore, a recent proteomics study in the hymenopteran Cardiocondyla obscurior 396

(Fuessl et al. 2014) yielded a few peptides mapping to the SPS1-UGA gene—albeit not 397

to the region including the UGA codon. Thus, we cannot unequivocally identify the amino 398

acid that is inserted in response to the UGA codon. Given that we observed two 399

independent UGA to Arg substitutions within insects, Arg could be a potential candidate. 400

Nonetheless, the recognition of a UGA codon by a standard tRNA for Arg would require 401

at best one mismatch in the first position of the codon (third position of 402

the anticodon), which is expected to compromise translation. 403

Functional hypothesis: parallel subfunctionalization generates SPS1 404

proteins 405

Even though originating from independent gene duplications of the same orthologous 406

gene (Figures 3 and 4), the pattern of strong sequence conservation suggests that SPS1 407

genes share a common function. It has been demonstrated for both insects and 408

vertebrate SPS1 that their function is different from SPS2 (Persson et al. 1997)(Xu et al. 409




14

2007a). We therefore suggest that the ancestral SPS2 protein at the root of metazoans 410

had not only its catalytic activity (i.e., synthesis of selenophosphate from selenide), but 411

also an additional, unknown function. Eventually, several metazoan lineages split these 412

two, with a new, duplicated protein, SPS1, assuming this other function. If this 413

hypothesis is true, then SPS1 proteins (although paraphyletic) should have similar 414

functions. To test this hypothesis we designed rescue experiments in Drosophila 415

melanogaster. The SPS1 mutation (SelDptuf ) is lethal in homozygous larvae and results 416

in very reduced and aberrant imaginal disc epithelia (Figure 7A; Alsina et al. 1998). 417

Thus, we used the Gal4-UAS system to activate different metazoan SPS1 genes in 418

SelDptuf mutants and tested whether the imaginal disc phenotype could be reverted. We 419

drove expression of either the human SPS1-Thr, the SPS-Gly isoform from Ciona 420

intestinalis SPS-ae, or the SPS1-UGA from Atta cephalotes (ant, hymenopteran), cloned 421

downstream UAS sequences, using the ubiquitous armadillo-Gal4 driver (Supplementary 422

Material S7, Supplementary Figure SM7.1). We focused on the wing imaginal disc and 423

observed significant rescue using the SPS-Gly isoform from C.intestinalis, both in size 424

and shape (Figure 7C) and partial rescue in size when using the ant SPS1-UGA or the 425

human SPS1-Thr (Figures 7D, E). These experiments suggest that the tested 426

heterologous SPS1 proteins have a similar molecular function, which is, as previously 427

noted, distinct from selenophosphate synthesis. 428

Unequal selective pressure on SPS1 and SPS2 genes after 429

duplication 430

We observed substantial differences in the rate of non-synonymous vs synonymous 431

substitution (Ka/Ks) across metazoan SPS genes, between the branches corresponding 432

to SPS1 and SPS2 after duplication (see Supplementary Material S5). Thanks to the 433

large number of SPS sequences available, we could reliably quantify Ka/Ks for SPS1 and 434

SPS2 in vertebrates and insects, and also for their closest outgroups which did not 435

duplicate SPS (Supplementary Figures SM5.1 and SM5.2). For annelids and ascidians, 436

with fewer sequences available, we obtained less accurate estimates (Supplementary 437

Figure SM5.3). After duplication, SPS2 genes display much higher values of Ka/Ks than 438

SPS1. The values for the unduplicated SPS2 genes are very similar to SPS1, and thus 439

lower than SPS2 after duplication. Although to different degrees, the trend is observed 440

for all SPS duplications in metazoans. It is evident both when comparing the extant 441

SPS1 and SPS2 sequences with the predicted ancestral sequence before duplication, 442




15

and when comparing extant sequences within the same orthologous group 443

(Supplementary Material SM5). This results in higher sequence divergence of the 444

duplicated SPS2 proteins with respect to SPS1, and also to SPS2 prior to duplication. 445

Indeed, SPS1 genes show overall higher protein sequence similarity across metazoans 446

than SPS2 genes after duplication (e.g. 80% vs 70% for the sequence set in 447

Supplementary Figure SM5.3). Moreover, the extant unduplicated SPS2 genes exhibit 448

stronger sequence similarity to SPS1 genes, than to SPS2 genes after duplication. The 449

diverse rate of protein evolution after duplication posed a major challenge for 450

phylogenetic reconstruction, resulting in an artifact known as long branch attraction (see 451

Supplementary Material S3). The Ka/Ks difference in SPS1 vs SPS2 is particularly 452

pronounced for insects, likely connected to the documented degeneration of the Sec trait 453

in this lineage (Chapple and Guigó 2008). 454

455

Discussion 456

Gene duplications are generally assumed to be the major evolutionary forces generating 457

new biological functions (Lynch and Conery 2000). However, the mechanisms by means 458

of which duplicated gene copies are maintained and evolve new functions are still poorly 459

understood (Innan and Kondrashov 2010). First, the evolutionary history of genes is 460

inherently difficult to reconstruct, since numerous factors deteriorate and confound 461

phylogenetic signals (Philippe et al. 2011). Second, assigning functions to genes is 462

difficult. Even the very same concept of function is controversial, and there is no 463

universal definition of what constitutes function (Kellis et al. 2014). From a pragmatic 464

standpoint, function is often equated to some sort of biochemical activity, which, in turn, 465

is intimately connected to specific experiments to measure it. But this fails to grasp that 466

genes work in a concerted way within a given biological context, and thus, even similar 467

biochemical activities may result in very different biological functions (phenotypes). Also, 468

experimental validation of function is possible only for a few genes, while for the great 469

majority, function is solely assigned by inference based on sequence similarity. 470

However, there are no universal thresholds to discriminate different functions from levels 471

of sequence divergence, since cases of exist of conserved function with low sequence 472

similarity and conversely, different functions can be carried out by highly similar 473

sequences. For all these reasons, functional evolution of genes is particularly difficult to 474

trace. 475




16

Here, we have investigated the evolutionary history of selenophosphate synthetase 476

genes (SelD/SPS) along the entire tree of life. SPS constitutes a particularly appropriate 477

gene family to investigate evolution of function. Since it is required for selenoprotein 478

synthesis, we can monitor its function as a genomic phenotype, by inspecting genomes 479

for selenoproteins and for other gene markers of selenium utilization. Moreover, SPS is 480

unique among all genes in that it is part of the Sec machinery and it is often a 481

selenoprotein itself. As a selenoenzyme, we presume that most likely its UGA-encoded 482

Sec residue can be functionally replaced only with Cys, and we thus expect that 483

mutations to codons other than Cys would impair its selenophosphate synthetase 484

function. 485

Consistent with this, we have found that Cys- or Sec- containing SPS genes (which we 486

refer as SPS2) are found in all genomes encoding selenoproteins, while those metazoan 487

genomes containing only SPS genes carrying amino acids other than Sec or Cys at the 488

homologous UGA position, do not encode selenoproteins. Our results suggest that the 489

non-Cys, non-Sec SPS genes share a common function, likely unrelated to 490

selenophosphate synthesis, and we refer collectively to them as SPS1. In SPS genes, 491

thus, the amino acid inserted at this single codon position seems to determine 492

unequivocally protein function, and is intertwined to the evolutionary fate of an entire 493

class of proteins (selenoproteins). Thanks to this feature, maybe unique among all 494

protein families, we have been able to untangle the complex functional evolution of SPS 495

genes, in particular within metazoans, where it involves a series of independent gene 496

duplications and the associated subfunctionalization events, from an ancestral Sec 497

carrying SPS gene that had both the SPS1 and SPS2 functions. In some ascidians, the 498

duplication originated by selective retro transposition of the SPS2 isoform of a single 499

gene encoding both SPS1 and SPS2 isoforms. Alternative transcript isoforms and gene 500

duplication are considered the main mechanisms contributing to increase protein 501

diversity. They anticorrelate at the genomic scale, so that protein families with many 502

paralogues tend to have fewer transcript variants, and vice versa (Talavera et al. 2007). 503

It is therefore expected that some genes shifted from one strategy to the other during 504

evolution: the function of an alternative isoform in one species would be carried by a 505

duplicated copy of the gene in another species. However, other than the ascidian SPS 506

genes reported here, only a handful of cases have been so far reported: the eukaryotic 507

splicing factor U2AF1 in vertebrates (Pacheco et al. 2004), the ey gene in Drosophila 508

(also known as Pax6)(Dominguez et al. 2004) and the mitf gene in fishes (Altschmied et 509




17

al. 2002). Very recently, Lambert et al. (2015) have suggested that this phenomenon 510

could be widespread during vertebrate evolution. 511

Gene duplication and alternative transcription differ in the efficiency with which they 512

separate functions. Duplications fully segregate the functions to different loci—allowing 513

both independent sequence evolution and independent regulation. In contrast, 514

alternative transcription/splicing isoforms typically share regions of identical sequence, 515

as well as, generally, proximal and distal regulatory regions. Thus, when a single gene 516

carries a dual function (even if exerted through alternative isoforms), certain sequence 517

segments are under two simultaneous sources of selection, which may be in 518

competition. Gene duplication and subfunctionalization provides a way to entirely escape 519

from the adaptive conflict (Hittinger and Carroll 2007). The fact that convergent 520

subfunctionalization duplications occurred several times independently within metazoans 521

is suggestive of selective advantage in having the two SPS functions carried out by 522

different genes. Here, we have detected duplications occurring at the root of vertebrates, 523

within tunicates, within annelids and at the root of insects. However the metazoan 524

genome space remains largely unexplored and other duplications are likely to be 525

uncovered in the future, as additional genome sequences become available. 526

Overall, the history of metazoan SPS genes contributes a striking example how function 527

evolves across orthologues and paralogues through a complex pattern of duplication 528

and loss events (Gabaldón and Koonin 2013). Specifically, it constitutes a prototypical 529

case of a particular “functional evolution path”: an ancestral state of dual/redundant 530

function, followed by subfunctionalization through gene duplication which occurs 531

independently in parallel lineages of descent. Analogous cases have been previously 532

reported (e.g. the β-catenin/armadillo gene during insect evolution (Bao et al. 2012)). 533

Parallel gene duplications result in a complex pattern of orthology/paralogy relationship 534

(Gabaldón 2008). Within monophyletic lineages with both SPS1 and SPS2 originated by 535

a duplication event (e.g. vertebrates), these genes are always paralogous, but both 536

genes are phylogenetically co-orthologous to SPS2 in lineages with a single SPS (e.g. 537

flatworms). Also, these genes are phylogenetically co-orthologous to both SPS1 and 538

SPS2 in other lineages with a distinct, parallel duplication event (e.g. insects). 539

Nonetheless, if we assume that subfunctionalization occurred in the same way in the 540

different lineages, then the SPS1 genes generated independently can be considered 541

functional orthologous, despite the lack of direct phylogenetic descent. In this view, it 542




18

may appear counter-intuitive that SPS1 genes have replaced the Sec/Cys site with 543

lineage-specific amino acids which are so dissimilar. This suggests that the residue 544

occurring at this particular position is irrelevant to SPS1 function. 545

After each duplication event documented here, the SPS2 gene increased the rate of 546

non-synonymous vs synonymous substitution (Ka/Ks). This can be interpreted as a 547

relaxation of the selective constraints acting on the coding sequence. In contrast, SPS1 548

genes preserve lower values of Ka/Ks, comparable to the parental gene before 549

duplication (Supplementary Material S5). It is remarkable that this trend is observed 550

consistently throughout all metazoan SPS duplications, despite the vast diversity in 551

duplication mechanisms. We must conclude that this evolutionary pattern is intimately 552

connected to the functions of the two SPS copies after duplication. Intuitively, the 553

relaxation of constraints in the SPS2 gene after duplication is well consistent with the 554

scenario of subfunctionalization that we propose. However, we may have expected the 555

duplicated SPS1 genes to also show relaxation compared to the parental gene before 556

duplication, since the dual-function state has presumably the highest selective 557

constraint. In contrast, the constraints acting on SPS1 after duplication are very similar 558

to those before duplication. These observations may suggest that the selective 559

constraints acting on the ancestral, dual-function SPS2 gene are imputable mainly to 560

selection for the SPS1 function, rather than for the canonical selenophosphate 561

synthetase activity. 562

Our results suggest that readthrough of the SPS UGA codon to incorporate amino acids 563

other than Sec played an important role in metazoan SPS duplication and 564

subfunctionalization. Indeed, we hypothesize that simultaneous to the production of 565

canonical Sec coding transcripts, selenoprotein genes (including SPS2) could produce 566

truncated SECIS-lacking mRNAs, due to inefficient transcription, or through a regulated 567

process, such as alternative usage of 3’ exons or poly-adenylation sites. In fact, 568

translational regulation is known to play an important role for many selenoproteins 569

(Howard et al. 2013). In at least one case (Selenoprotein S, VIMP gene), this is achieved 570

by the regulated inclusion/exclusion of the SECIS element in the mature transcripts 571

through alternative splicing (Bubenik et al. 2013). In such SECIS-lacking truncated 572

transcripts, no Sec insertion will take place, and termination of translation at the UGA 573

codon is the most likely outcome. However, if alternative non-SECIS mediated 574

readthrough mechanisms are present, other amino acids could still be incorporated in 575




19

response to the UGA codon. If this amino acid is other than Sec or Cys, the resulting 576

protein may not be able to perform its original function (as in SPS2/SPS1), but it may 577

develop a novel one. We speculate that this was the case for the ancestral metazoan 578

SPS gene, in which, possibly, the novel SPS1 function emerged in a secondary, non-579

Sec isoform maybe produced through regulated SECIS inclusion. The readthrough 580

enhancing stem loop structures around the UGA codon (SRE and HRE) found in SPS 581

genes support this hypothesis. These structures are particularly strong in the SECIS-582

lacking hymenopteran SPS1-UGA genes, which, in addition, contain the readthrough 583

enhancing hexanucleotide. Then, when the SPS gene duplicates, the SECIS is lost in 584

the SPS1 copy, leading to complete subfunctionalization. The whole process is best 585

seen within insects. Hymenoptera, after the SPS duplication, lost SPS2 when 586

selenoproteins disappeared from the genome, but still conserved SPS1-UGA. 587

Paraneopterans with selenoproteins (R.prolixus and P.humanus) still maintain the two 588

genes (SPS1 and SPS2) with UGA. In pea aphid and in non-hymenopteran 589

Endopterygota, the in-frame UGA in SPS1-UGA mutated to an Arg codon, becoming the 590

“standard” gene known as Drosophila SPS1. 591

It is tempting to speculate that the SRE/HRE stem loop structures, which are found not 592

only in SPS, but also in some other selenoprotein genes (Howard et al. 2005), are the 593

eukaryotic homologues of the bacterial SECIS element (bSECIS, see Supplementary 594

Figure SM6.1 and SM6.2). Indeed, the bacterial Sec insertion system is different from its 595

eukaryotic counterpart, both regarding the structure and the localization of the SECIS 596

element. In eukaryotes, the SECIS is characterized by a kink-turn core, and it is located 597

in the 3’ UTR. In bacteria, the bSECIS it is a simple stem-loop structure (lacking the kink-598

turn motif), located immediately downstream of the Sec-UGA within the coding 599

sequence. bSECIS elements are read by SelB, a Sec-specific elongation factor with a 600

specialized N-terminal domain. In eukaryotes, SECIS elements are bound by SECIS 601

binding protein 2 (SBP2), which recognizes specific structural features mainly around its 602

kink-turn core (Krol 2002). 603

The evolutionary history of the SECIS elements across the domains of life remains 604

largely unexplored. The assumption that SECIS and bSECIS are phylogenetically 605

homologous structures requires the re-location of the bSECIS to the 3’UTR, concomitant 606

with the radical alteration of its structure—for both of which it is difficult to postulate 607

plausible evolutionary mechanisms. In contrast, SRE/HRE are stem loop structures that 608




20

localize next to the UGA codon, and resemble much more the bSECIS structure than the 609

SECIS does. In addition to structural similarity, bSECIS and HRE/SRE also share 610

functional similarity, since both disfavor termination at Sec-UGA sites during translation. 611

Thus, we hypothesize that the SRE/HRE structures (at least those in SPS genes) are 612

derived from bSECIS, and that the eukaryotic SECIS is an evolutionary innovation 613

unconnected to the bSECIS. After the emergence of the eukaryotic SECIS system, the 614

ancestral bSECIS function was ”downgraded” to helper for Sec insertion (SRE). In 615

ancestral metazoans it was kept under selection to allow both Sec-insertion and non-Sec 616

readthrough, as the non-Sec isoform acquired the SPS1 function. Within insects, after 617

the SPS duplication, the ancestral bSECIS structure remained in one of the duplicated 618

copies (SPS1-UGA), specializing only in non-Sec readthrough. This structure has been 619

conserved in hymenopterans and some paraneopterans, becoming what we named here 620

HRE. 621

In summary, thanks to the singular feature that the amino acid occurring at a single 622

position serves as binary indicator of gene function, we traced the evolutionary history of 623

SelD/SPS genes with unprecedented detail, providing, at the same time, a survey of 624

selenium utilization traits across the entire tree of life. In metazoans, the SPS phylogeny 625

constitutes a prototypical case of functional evolution, in which dual function is 626

segregated to different loci through independent gene duplications and subsequent 627

convergent subfunctionalization. 628

629

Methods 630

Gene prediction 631

We performed gene prediction using Selenoprofiles ver. 3.0 (Mariotti and Guigó 2010) 632

(http://big.crg.cat/services/selenoprofiles). This program scans nucleotide sequences to 633

predict genes belonging to given protein families, provided as amino acid sequence 634

alignments (profiles). When searching for a selenoprotein family (at least one sequence 635

with a Sec residue), the program can find both selenoprotein genes and homologues 636

with other amino acids at this position, as long as their full protein sequence is similar 637

enough to the input profile alignment. To ensure maximum sensitivity, two manually 638

curated profiles were used for SPS, one containing sequences from all lineages and 639

another only from prokaryotes. Specificity was controlled through a set of filters applied 640




21

to predicted candidate genes, which include checking similarity to the profile sequences, 641

and best matches among all known proteins in the NCBI non-redundant (NR) database 642

(AWSI score and tag score – see Selenoprofiles manual). We built our SPS gene 643

datasets (available in Supplementary Material S7) searching a large collection of 644

eukaryotic (505) and prokaryotic genomes (8.263 with a non redundant reference subset 645

of 223), downloaded mainly from NCBI. For eukaryotes and for prokaryotes in the 646

reference set, results were manually inspected and filtered to exclude duplicates, 647

pseudogenes (abundant in vertebrates) and contaminations of the genome assemblies. 648

Eukaryotic SECIS elements were searched using the program SECISearch3 (Mariotti et 649

al. 2013). 650

We also used Selenoprofiles with profiles derived from protein families that are markers 651

for other selenium utilization traits, ybbB and SelA. We used the same program with a 652

comprehensive collection of selenoprotein families in a semi-automatic procedure to 653

probe the number of selenoproteins per lineage (as those displayed in Figures 1 and 2). 654

tRNAscan-SE ver. 1.23 (Lowe and Eddy 1997) and Aragorn ver. 1.2.28 (Laslett and 655

Canback 2004) were used to search for tRNAsec. We noticed the presence of abundant 656

false positives in prokaryotes, lacking the long extra-arm characteristic of tRNAsec. 657

Thus, we focused most of our analysis on the reference set, inspecting manually 658

candidates and filtering out all such cases. 659

For ciliates all predictions were manually adjusted, given their non-standard genetic 660

code. In addition to genomes, the NCBI EST database was also used to investigate 661

certain eukaryotic lineages of interest, such as tunicates, annelids and birds 662

(Supplementary Materials S3 and S4). 663

Phylogenetic analysis 664

Alignments were computed using T-Coffee ver. 8.95 (Notredame et al. 2000) and 665

sometimes complemented by MAFFT ver. 7.017b (Katoh et al. 2005). To deduce the 666

phylogenetic history of SPS, we combined three types of information: topology of gene 667

trees reconstructed from protein sequences, phylogenetic tree of investigated species, 668

and positions of introns in respect to protein sequence. Gene trees were computed by 669

maximum likelihood as explained in (Mariotti et al. 2012) after (Huerta-Cepas et al. 670

2011), and visualized using the program ETE ver. 2 (Huerta-Cepas et al. 2010). The 671

approximate phylogenetic tree of investigated species was derived from the NCBI 672

taxonomy database (Sayers et al. 2009), and was refined for insects with data from The 673




22

International Aphid Genomics Consortium (2010). Figures 1, 2 and Supplementary 674

Figure SM1.1 were generated with the R package ggsunburst, available at 675

http://genome.crg.es/~didac/ggsunburst/. Relative positions of introns were visualized 676

using selenoprofiles_tree_drawer (available within Selenoprofiles) and ETE. 677

The SPS phylogenetic history presented here has been deduced using parsimony as the 678

main driving principle. Supplementary Material S3 contains a detailed description of the 679

process to solve the phylogeny of eukaryotic SPS. Supplementary Material S4 is 680

dedicated to SPS genes in tunicates. 681

Evolutionary analysis 682

We analyzed the ratio of non-synonymous vs synonymous substitution rates (Ka/Ks) in 683

metazoan SPS genes using the program Pycodeml (Mariotti, unpublished) available in 684

Supplementary Material S8, or online at https://github.com/marco-mariotti/pycodeml. 685

This program runs internally CodeML, a program which is part of the PAML package 686

(Yang 2007) to predict the sequence at ancestral nodes. Then, it computes various 687

indexes of sequence evolution related to Ka/Ks, fully explained in Supplementary Material 688

S5. Pycodeml can also produce a graphical output including the sequence alignment, 689

allowing to inspect in detail any substitution (available at http://big.crg.cat/SPS). We 690

have run Pycodeml on three manually curated alignments of coding sequences: one for 691

the insect SPS duplication (Supplementary Figure SM5.1), one for the vertebrate SPS 692

duplication (Supplementary Figure SM5.2), and one “summary set” containing 693

representatives for all four SPS duplications here described (Supplementary Figure 694

SM5.3). Each alignment was trimmed manually to leave out the N-terminal and C-695

terminal tails, and to remove any insertion that occurs only in any single sequence. 696

Supplementary Material S5 contains the results of the evolutionary analysis, and an 697

explanation of all sequence statistics applied. 698

Detection of extensions and fusions 699

We used two different strategies to detect additional domains in SPS genes. First, we 700

searched for annotated SPS fusions. We ran our SPS gene set with BLASTp (Altschul et 701

al. 1997) against the NCBI NR database. Then we parsed the results, searching for 702

large stretches of sequence of a matched NR protein that were not included in the output 703

BLAST alignment. Second, we looked in genomes for any SPS extension. We expanded 704

each predicted SPS gene at both sides until a stop codon was reached, and we ran the 705




23

extensions with BLASTp against NCBI NR database. All candidates from the two 706

methods were merged, clustered by similarity, and manually inspected. Conservation in 707

multiple species and confirmation with RNA data were used as criteria to exclude 708

artifacts possibly caused by our detection method or by imperfect genome assemblies, 709

thus prioritizing specificity over sensitivity. For the most interesting cases, a new 710

alignment profile was built including the protein sequence of SPS and of the additional 711

domain, and used to search again the genome sequences. Supplementary Material S2 712

contains a description of results. 713

Prediction of conserved secondary structures 714

The program RNAz ver. 2.1 (Gruber et al. 2010) was used to predict conserved 715

secondary structures embedded in SPS coding sequences. Initially, we produced a 716

“master alignment” that included the coding sequences of all SPS genes in our dataset. 717

The nucleotide sequence alignments employed were based on the alignment of the 718

corresponding amino acid sequences. Then, the master alignment was used to extract a 719

multitude of “subset alignments”, which included only genes in specific lineages, and/or 720

only specific types of SPS (based on the residue found at the Sec position). RNAz was 721

run either directly on these subset alignments, or instead the program trimAl ver. 1.4 722

(Capella-Gutiérrez et al. 2009) was used in advance to reduce the number of 723

sequences. All secondary structures predicted in this way were then manually inspected. 724

For the best candidates, images of consensus structures were generated using the 725

ViennaRNA package (Lorenz et al. 2011)(see Supplementary Figures SM6.1 and 726

SM6.2). Supplementary Material S6 contains a detailed description of the procedure and 727

of the results, including the list of subset alignments considered. 728

Rescue experiments in Drosophila 729

For rescue experiments we used the Gal4/UAS system (Brand and Perrimon 1993). We 730

obtained cDNA for human SPS1 from the Harvard resource core 731

(http://plasmid.med.harvard.edu/PLASMID/). For SPS-ae of C.intestinalis, we obtained 732

the cDNA corresponding to the Gly isoform by performing targeted PCR on larvae 733

extracts. We obtained cDNA for SPS1-UGA from A.cephalotes by performing targeted 734

PCR on extracts provided by James F.A. Traniello. These cDNAs were cloned through 735

the Gibson method (Gibson et al. 2009) into the pUAST-attB vector linearized by double 736

digestion with BglII and XhoI and clones verified by Sanger sequencing. Primers used 737

for cloning are reported in Supplementary Material S7. Transgenic flies were obtained 738




24

following the method described by (Bischof et al. 2007). Line yw M{e.vas-int.DM}ZH-2A; 739

3: M{RFP.attP}ZH-86Fb was used to direct the insertion into the 3R chromosome (86F). 740

Crosses were designed to obtain expression of each transgenic SPS1 into a SelDptuf 741

homozygous mutant background (Supplementary Material S7, Supplementary Figure 742

SM7.1). We used armadillo-Gal4 (arm-Gal4) as a driver to activate expression of the 743

UAS-cDNA inserts. The final cross was: SelDptuf/CyOdfYFP; UAS-cDNA insert/MKRS x 744

SelDptuf/CyOdfYFP; arm-Gal4/TM6B. Expression of transgenes under arm-Gal4 745

promoter was confirmed by RT-PCR (Supplementary Figure SM7.2). Imaginal wing 746

discs from third instar larvae were dissected in PBS and stained with Rhodamine 747

Phalloidin from Molecular Probes® (cat #R415). 748

Acknowledgements 749

We thank James F.A. Traniello and Ysabel Milton Giraldo (Boston University, Boston) for 750

providing extracts of A.cephalotes. We thank the Drosophila Injection Platform of the 751

Consolider Project (CBM-SO, Madrid) for production of transgenic flies. RG group 752

research was funded by grants BIO2011-26205 from the Spanish Ministry of Science 753

and grant SGR-1430 from the Catalan Government. MM received a FPU doctoral 754

fellowship AP2008-04334 from the Spanish Ministry of Education. TG group research 755

was funded in part by a grant from the Spanish Ministry of Economy and 756

Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund 757

grant (NPRP 5-298-3-086), and a grant from the European Research Council under the 758

European Union's Seventh Framework Programme (FP/2007-2013) / ERC (Grant 759

Agreement n. ERC-2012-StG-310325). VNG group research was supported by NIH 760

GM061603. 761

Disclosure declaration 762

Authors declare no conflict of interest. 763

Figure legends 764

Figure 1: Phylogenetic profile of SPS and selenium utilization traits in 765

prokaryotes. The sunburst tree shows the phylogenetic structure of the reference set of 766

223 prokaryotic genomes (taken from NCBI taxonomy), and the presence of SelD genes 767

and other markers of selenium utilization. The section for archaea is zoomed in as a 768

guide to interpret the plot (1). Every ring-shaped section represents a taxonomic rank in 769




25

NCBI taxonomy (superkingdom, phylum, class, order, family, genus). The last two 770

outermost rings display features for each of the species analyzed. The length of the 771

black bars protruding from the outermost circle is proportional to the number of 772

selenoproteins detected in each species. The outermost ring is color coded for the 773

presence of ybbB and SelA in the species, with a black dot inside for denoting tRNAsec 774

presence. The second outermost ring is labeled for SelD presence and type: “SelD-Sec”, 775

“SelD-Cys”, “no gene found”. The color is propagated to the lower ranks by hierarchy. 776

Transparency is used to display how many species under a lineage have the same label. 777

Assuming no Cys to Sec conversion and no horizontal transfer, colors reflect the 778

predicted SelD presence at ancestral nodes. This allows detecting the Sec to Cys 779

conversions manually, for example in Clostridia (2). The hierarchical color assignment is 780

violated only in the case of Gammaproteobacteria, altered to be red. In fact, although its 781

sublineage Pasteurellales contains SelD-Sec (see 3), our analysis points to a Cys to Sec 782

conversion instead, which implies that the ancestral state for Gammaproteobacteria was 783

SelD-Cys. The plot can be used to map the Sec and selenouridine (SeU) traits (see top-784

right panel). For example Escherichia coli (4) has both traits, Pasteurellales only Sec (3), 785

and Bacillus coagulans only SeU (5). Enterococcus faecalis has a SelD-Cys gene, but 786

no other selenium utilization marker (6) (Romero et al. 2005)(Zhang et al. 2008). 787

Expanded versions of the plot (up to 8,263 species) are available in Supplementary 788

Material S1. Note that gene fusions and extensions are not represented in this plot. 789

Figure 2: Phylogenetic profile of SPS genes and approximate selenoproteome size 790

of eukaryotes. The plot recapitulates the results on 505 genomes analyzed, 791

summarized to 213 displayed here. The tree (from NCBI taxonomy) is partitioned in 792

lineages, highlighted in grey tones to help visualization. Near the tips of leaves, the 793

presence of SPS proteins is displayed using colored rectangles. Sec (green) and Cys 794

(red) forms correspond to SPS2 (top left legend), while the other homologues to SPS1 795

(top right legend). The gene extensions found for some SPS2-Cys are indicated with 796

symbols inside the rectangle (top left legend). The number of selenoproteins predicted in 797

each genome is indicated with a black vertical bar extending from the tips. As reference 798

points, mammals have ~25 selenoprotein genes, D.melanogaster has three, and 799

C.elegans only one. 800

Figure 3: Parallel gene duplications of SPS proteins in metazoa. The plot 801

summarizes the phylogenetic history of metazoan SPS genes, consisting of parallel and 802




26

convergent events of gene duplication followed by subfunctionalization. Each colored 803

ball represents a SPS gene, indicating the residue found at the UGA or homologous 804

codon (U for selenocysteine, C for cysteine, T for threonine, G for glycine, L for leucine, 805

R for arginine, x for unknown residue). The gene structures are schematically displayed 806

in Figure 4. The names of the insect species lacking selenoproteins are in red. The main 807

genomic events shaping SPS genes are indicated on the branches: GD whole gene 808

duplication, GDR gene duplication by retro-transposition, AE origin of an alternative 809

exon, SL Sec loss, SC conversion of Sec to Cys, SO conversion of Sec to something 810

other than Cys, GL gene loss. In our subfunctionalization hypothesis (see text), we map 811

the origin of a dual function at the root of metazoa. 812

Figure 4: Structure and function of the identified SPS genes. SPS proteins are 813

classified according to the residue found at the UGA or homologous position (see also 814

Figure 3). The presence of specific secondary structures is also indicated: bSECIS for 815

bacterial SECIS element, SRE for Sec recoding element (Howard et al. 2005), SECIS for 816

eukaryotic SECIS element, HRE for hymenopteran readthrough element. The rightmost 817

column indicates the functions predicted for the SPS proteins. SPS2 function is the 818

synthesis of selenophosphate. SPS1 function is defined as the uncharacterized 819

molecular function of Drosophila SPS1-Arg (double underlined), which is likely to be 820

similar to that of other SPS1 genes, as suggested by KO-rescue experiments in 821

Drosophila (underlined). *: for eukaryotic SPS2, the parentheses indicate that some such 822

genes are predicted to possess both SPS1 and SPS2 functions, those marked also with 823

a star (*) in Figure 3 (basically all metazoans with no SPS1 protein in the same 824

genome). 825

Figure 5: Alternative SPS1/2 transcript isoforms sorted by retrotransposition 826

within ascidians. This figure expands the section for tunicates in Figure 3. At the root of 827

ascidians, the ancestral SPS2-Sec gene acquired a novel SPS1-Gly transcript isoform, 828

through alternative exon usage at the 5’ end (AE). Then, at the root of the ascidian 829

lineage Styelidae and Pyuridae, the SPS2-Sec transcript of this dual SPS1/SPS2 gene 830

(SPS-ae) retrotransposed to the genome creating a novel SPS2-Sec gene (GDR). This 831

presumably triggered the loss of Sec from the parental gene which, as both the SECIS 832

and the UGA containing exon degenerated (SL), specialized only in the production of 833

SPS1-Gly. 834

Figure 6: Readthrough-enhancing hexanucleotide in SPS genes. The phylogenetic 835




27

tree on the left shows the nucleotide sequence alignment at the UGA (or homologous) 836

site in SPS sequences. Only SPS2 and SPS1-UGA genes are shown here. Codons are 837

colored according to their translation, following the same color schema used in Figures 2 838

and 4 (grey for other amino acids). The presence of the hexanucleotide described in 839

(Harrell et al. 2002) is marked with a black dot. Green dots mark the genes for which a 840

bona-fide SECIS element was identified. The last column indicates the presence of 841

SPS1 genes. 842

Figure 7: Rescue of the Drosophila melanogaster SPS1 mutant by heterologous 843

SPS1 proteins. All images show wing imaginal discs dissected from larvae with the 844

indicated constructs and genotypes. (A,B) The SPS1 mutant flies (SelDptuf / SelDptuf ) 845

result in defects in the whole organism, but can be easily monitored in the wing imaginal 846

disc epithelia; the homozygous condition (A) strongly impairs size and morphology, 847

whereas the heterozygous (B) is very similar to the wild type condition. (C-E) The severe 848

homozygous SelDptuf / SelDptuf phenotype is partially rescued by ubiquitous expression 849

of heterologous SPS1 proteins. (C) SelDptuf / SelDptuf mutants with C. intestinalis SPS1-850

Gly. (D) SelDptuf / SelDptuf mutants with A.cephalotes SPS1-UGA. 851

(E) SelDptuf / SelDptuf mutants with human SPS1-Thr. Scale bars 50 µm. 852

853

Figures: 854

(attached) 855

856

Supplementary Material 857

Find next the following supplementary sections: 858

• S1: SelD in prokaryotes 859

• S2: Gene fusions and extensions 860

• S3: Phylogeny of eukaryotic SPS proteins 861

• S4: Alternative isoforms sorted by gene duplication in ascidians 862

• S5: Evolutionary analysis of metazoan SPS genes 863

• S6: Secondary structures within coding sequences of SPS genes 864

• S7: Rescue experiments in Drosophila 865




28

• S8: Datasets 866

867

868

869

870

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