Evolution of the Schlafen genes, a gene family associated with embryonic lethality, meiotic drive, immune processes and orthopoxvirus virulence

Abstract

Genes of the Schlafen family, first discovered in mouse, are expressed in hematopoietic cells and are involved in immune processes. Previous results showed that they are candidate genes for two major phenomena: meiotic drive and embryonic lethality (DDK syndrome). However, these genes remain poorly understood, mostly due to the limitations imposed by their similarity, close location and the potential functional redundancy of the gene family members. Here we use genomic and phylogenetic studies to investigate the evolution and role of this family of genes. Our results show that the Schlafen family is widely distributed in mammals, where we recognize four major clades that experienced lineage-specific expansions or contractions in various orders, including primates and rodents. In addition, we identified members of the Schlafen family in Chondrichthyes and Amphibia, indicating an ancient origin of these genes. We find evidence that positive selection has acted on many Schlafen genes. Moreover, our analyses indicate that a member of the Schlafen family was horizontally transferred from murine rodents to orthopoxviruses, where it is hypothesized to play a role in allowing the virus to survive host immune defense mechanisms. The functional relevance of the viral Schlafen sequences is further underscored by our finding that they are evolving under purifying selection. This is of particular importance, since orthopoxviruses infect mammals and include variola, the causative agent of smallpox, and monkeypox, an emerging virus of great concern for human health.

Fig. 1

SLFN proteins in mouse. The location of the common core region analyzed in our phylogenetic study is shown, as well as the AAA_4 (Pfam04326) and 52540 superfamily domains (P-loop containing nucleoside triphosphate hydrolases superfamily) (www.ensembl.org; Appendix A). The latter domain is present in DNA and RNA helicases and some other proteins. Slfn14 and LOC435271 are predicted genes without sufficient expression support (Table 1 and Appendix C) and, therefore, their products have not been represented. An alternative Slfn10 translation initiation site is indicated with a dashed line.

Fig. 2

Orthologous regions in mouse, rat, human and opossum that contain the Slfn genes. Indicated are the Ensembl annotated Slfn genes and the related sequences found in our search, their exons, and direction of transcription when expression support was found (Appendix A). The location of the sequences identified and used in our phylogenetic analyses (Fig. 4, Fig. 5) is indicated below with arrows. Colors indicate their relationships according to our phylogenetic results (red = Group 1; yellow = Group 2; blue = Group 3; green = “Group 4”; black = unidentified). The position of the human Slfn12L predicted transcript is indicated, although the dashed arrow box also encompasses several additional sequences that are not included in the prediction but that we subsequently identified as Slfns (Appendix A). Opossum R2 sequence was not included in the phylogenetic analysis (see Methods), but our analyses indicate that it is more similar to Slfn8, 9 and 10 (Group 4) sequences than to any other mouse sequences (data not shown).

Fig. 3

Distribution of Schlafen sequences among taxa. The topology and divergence times of the mammalian tree are drawn according to www.tolweb.org/tree and Murphy et al. (2007). The total numbers of Slfn sequences for each taxon are indicated, as well as the number of Slfn sequences that fell within each of the four groups identified in our phylogenetic analyses (Fig. 4, Fig. 5). Only sequences that were suitable for our phylogenetic analyses (see Methods) could be classified into one of the 4 groups. Genome sequencing status at the time of this analysis: ⁎2× (or less) whole genome shotgun; ^†underway, sequencing projects at early stages.

Fig. 4

Phylogeny of Slfn genes based on neighbor-joining analysis of amino acid sequences. Only sequences that aligned unambiguously to the mouse SLFN core region (Fig. 1) were analyzed. This is an unrooted phylogram obtained using the “equal input” model implemented in MEGA 3.1 (Kumar et al., 2004). We designate four major clades ”Groups 1–4”. The pink arrow indicates the hypothesized occurrence of horizontal transfer of a Slfn gene from a rodent to the OPV ancestor. Numbers at nodes represent bootstrap support values based on 5000 pseudoreplicates. ⁎ indicates 70% or above bootstrap support (Hillis and Bull 1993). Values below 50% were removed. Scale: 0.1 indicates 10% estimated number of substitutions per position.

Fig. 5

Phylogeny of Slfn genes based on Bayesian analysis of amino acid sequences. The tree is rooted using the sequence from “elephant fish” (chondrichthyan). Numbers at nodes represent posterior probabilities. Groups 1–4 (designated based on the unrooted neighbor-joining tree, Fig. 4) are indicated. Results from 4.5 million generations run under the “mixed” (multiple model) criterion for evolution are shown.

Fig. 6

v-slfn sequences in orthopoxviruses. A representative strain of each OPV species is depicted. v-slfn and surrounding genes are annotated according to www.poxvirus.org. v-slfn is flanked by sequences that code for a serine/threonine kinase and an ankyrin protein in all OPVs. The predicted v-slfn ORFs (www.poxvirus.com) often include non-Slfn sequences that code for a baculovirus p26 protein domain. The v-slfn sequences of all OPVs (Appendix D), as well as all the other sequences represented, were identified by BLAST search by using all mouse SLFN and the camelpox sequences as queries, respectively.