Structural representation and identification of positively-selected branches and codons in mammalian LGP2.

<p>(A) Based on human protein structure, the key domains of LGP2 (<a href="http://www.uniprot.org/uniprot/q96c10" target="_blank">http://www.uniprot.org/uniprot/Q96C10</a>) and the corresponding boundaries are schematically represented. Also, the human domain boundaries while in the mammalian LGP2 deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s006" target="_blank">Figure S6</a>) are shown in brackets. (B) Cladogram of 30 mammalian LGP2 genes collected from Ensembl and NCBI databases. Branch-site analyses were performed to identify specific branches under episodic positive selection. Branches with statistically significant likelihood ratio tests (LRTs) when performing PAML branch-site model A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t004" target="_blank">Table 4</a>) are colored in green; branch colored in blue has been simultaneously identified by PAML branch-site model A and Hyphy branch-site REL method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t005" target="_blank">Table 5</a>). (C) Positively-selected codons are exhibited in the table and numbered according to the mammalian LGP2 deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s006" target="_blank">Figure S6</a>). Colors on the codon numbering row correspond to the LGP2 domain with the same color in the protein structural representation (A). The background colors on the identified codons match different amino acid properties: polar positive (yellow), polar negative (orange), polar neutral (green), non-polar neutral (purple), non-polar aliphatic (blue) and non-polar aromatic (pink). The used abbreviations correspond, by order of appearance, to the following species: Hosa – Human; Patr – Chimpanzee; Papa – Bonobo; Gogo – Gorilla; Poab – Orangutan; Mamu – Rhesus macaque; Sabo – Black-capped squirrel monkey; Caja – Marmoset; Mimu – Mouse lemur; Otga – Bushbaby; Bota – Cow; Ovar – Sheep; Susc – Pig; Tutr – Dolphin; Mylu – Little brown myotis; Ptva – Large flying fox; Ptal – Black flying fox; Loaf – Elephant; Mupu – Ferret; Aime – Giant panda; Calu – Dog; Feca – Cat; Eqca – Horse; Ocpr – American pika; Orcu – European rabbit; Ictr – Squirrel; Crgr – Chinese hamster; Mumu – Mouse; Rano – Rat; Capo – Guinea pig. </p

Positive Evolutionary Selection On the RIG-I-Like Receptor Genes in Mammals

<div><p>The mammalian RIG-I-like receptors, RIG-I, MDA5 and LGP2, are a family of DExD/H box RNA helicases responsible for the cytoplasmic detection of viral RNA. These receptors detect a variety of RNA viruses, or DNA viruses that express unusual RNA species, many of which are responsible for a great number of severe and lethal diseases. Host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. Using six codon-based Maximum Likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. The highest number of positively selected codons was detected in MDA5, but a great percentage of these codons were located outside of the currently defined protein domains for MDA5, which likely reflects the imposition of both functional and structural constraints. Additionally, our results support LGP2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. On the other hand, the preponderance of positively selected codons for RIG-I were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this RNA helicase. Furthermore, the RIG-I repressor domain, the region responsible for recognizing and binding to its RNA substrates, exhibited the strongest evidence of selective pressures. Branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. In conclusion, by looking for evidence of positive evolutionary selection on mammalian RIG-I-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the RIG-I gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens. </p> </div

The remnant of the European rabbit (<i>Oryctolagus cuniculus</i>) IgD gene

<div><p>Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (<i>Cδ</i>) gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit <i>Cδ</i> gene approximately 21 kb downstream of the IgM gene. The remnant <i>Cδ</i> locus lacks the <i>δCH1</i> and hinge exons, but contains truncated <i>δCH2</i> and <i>δCH</i>3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the <i>Cδ</i> gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.</p></div

Maximum likelihood (ML) phylogenetic trees of mammalian MDA5 gene used for codon-based ML analysis.

<p>When testing mammalian MDA5 alignment for recombination, one significant breakpoint was detected at nucleotide position 903. (A) A phylogenetic tree was reconstructed for the first 903 nucleotides under the nucleotide substitution model TIM3+G. (B) A second ML tree was inferred for the remaining 2211 nucleotides and under the nucleotide substitution model TIM3+I+G. (C) A tree was also reconstructed for MDA5 total alignment without recombination testing and under the nucleotide substitution model GTR+G. Bootstrap values >50 are indicated on the branches. </p

Maximum likelihood phylogenetic trees for mammalian <i>Cδ</i> exons.

<p>A) <i>δCH2</i>, B) <i>δCH3</i>, C) <i>Cδ</i> secretory exon, D) <i>Cδ</i> transmembrane exon 1. Platypus included as an outgroup in D. Bootstrap values from 1000 replicates appear next to the nodes.</p

Rabbit remnant <i>Cδ</i> transmembrane (<i>δTM1</i> and <i>δTM2</i>) exons.

<p>A) Nucleotide sequence alignment of rabbit remnant <i>δTM1</i> exon with <i>δTM1</i> exons of various mammalian species. Potential 5’ and 3’ splice sites in rabbit <i>δTM1</i> enclosed in boxes. B) Amino acid sequence alignment of rabbit and other mammalian <i>δTM1</i> exons. RF1 and RF2 indicate reading frames 1 and 2 for rabbit <i>δTM1</i>. Asterisks indicate stop codons. C) Nucleotide sequence alignment of rabbit remnant <i>δTM2</i> exon with <i>δTM2</i> exons of various mammalian species. Potential 5’ splice site in rabbit <i>δTM2</i> enclosed in box. Translated region in A) and C) indicated by uppercase letters. dashes, nucleotide or amino acid gaps.</p

Structural representation and identification of positively-selected branches and codons in mammalian RIG-I.

<p>(A) Based on human protein structure, the key domains of RIG-I (<a href="http://www.uniprot.org/uniprot/o95786" target="_blank">http://www.uniprot.org/uniprot/O95786</a>) and the corresponding boundaries are schematically represented. Also, the human domain boundaries while in the mammalian RIG-I deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s004" target="_blank">Figure S4</a>) are shown in brackets. (B) Cladogram of 26 mammalian RIG-I genes collected from Ensembl and NCBI databases. Branch-site analyses were performed to identify specific branches under episodic positive selection. Branches with statistically significant likelihood ratio tests (LRTs) when performing PAML branch-site model A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t002" target="_blank">Table 2</a>) are colored in green; branches simultaneously identified by PAML branch-site model A and Hyphy branch-site REL method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone-0081864-t005" target="_blank">Table 5</a>) are colored in blue. (C) Positively-selected codons are exhibited in the table and numbered according to the mammalian RIG-I deduced protein sequences alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081864#pone.0081864.s004" target="_blank">Figure S4</a>). Symbol “-” represents a deletion. Colors on the codon numbering row correspond to the RIG-I domain with the same color in the protein structural representation (A). The background colors on the identified sites match different amino acid properties: polar positive (yellow), polar negative (orange), polar neutral (green), non-polar neutral (purple), non-polar aliphatic (blue) and non-polar aromatic (pink). The used abbreviations correspond, by order of appearance, to the following species: Hosa – Human; Patr – Chimpanzee; Papa – Bonobo; Gogo – Gorilla; Poab – Orangutan; Paan – Olive baboon; Mamu – Rhesus macaque; Sabo – Black-capped squirrel monkey; Caja – Marmoset; Mimu – Mouse lemur; Otga – Bushbaby; Bota – Cow; Ovar – Sheep; Susc – Pig; Mylu – Little brown myotis; Ptva – Large flying fox; Ptal – Black flying fox; Aime – Giant panda; Calu – Dog; Feca – Cat; Eqca – Horse; Loaf – Elephant; Ictr – Squirrel; Capo – Guinea pig; Mumu – Mouse; Orcu – European rabbit. </p

Nucleotide positions of rabbit remnant <i>Cδ</i> exons and neighboring DNA elements in BAC clone 27N5.

<p>Nucleotide positions of rabbit remnant <i>Cδ</i> exons and neighboring DNA elements in BAC clone 27N5.</p

Maximum likelihood (ML) phylogenetic tree of RIG-I gene used for codon-based ML analysis.

<p>The GTR+G nucleotide substitution model was employed in mammalian RIG-I gene phylogenetic reconstruction. Bootstrap values >50 are indicated on the branches.</p

The remnant of the rabbit IgD locus.

<p>The remnant of the rabbit <i>Cδ</i> gene and the inserted repetitive DNA elements downstream of the <i>Cμ</i> transmembrane exons are depicted. The rabbit <i>Cδ</i> exons are represented by black rectangles. White rectangles adjacent to the <i>δCH2</i> and <i>δCH3</i> exons indicate regions of loss of similarity to their homologs in other mammalian species (the non-homologous region of <i>δCH2</i> overlaps L1MA9 5). Repetitive DNA elements are labeled and designated by colored rectangles. The directionality of C repeats, and direct repeats flanking the 5’-most endogenous retrovirus, are indicated by arrowheads. The putative σδ switch region is indicated by a hatched rectangle. <i>δS</i>, <i>Cδ</i> secretory exon; <i>δTM1</i>, <i>Cδ</i> transmembrane exon 1; <i>δTM2</i>, <i>Cδ</i> transmembrane exon 2; <i>μTM1</i>, <i>Cμ</i> transmembrane exon 1; <i>μTM2</i>, <i>Cμ</i> transmembrane exon 2; ψHMG14, ψASB, ψMAN, high-mobility group 14, arylsulfatase B and endo-alpha-like mannosidase processed pseudogenes, respectively; σδ, putative σδ switch region; nomenclature for C repeats (and the LINE1, L1OcμM) follows that adopted in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182029#pone.0182029.ref007" target="_blank">7</a>] (C13 was not present in the genomic DNA used in that study); nomenclature of other repetitive DNA elements follows that used by the Genetic Information Research Institute [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182029#pone.0182029.ref032" target="_blank">32</a>].</p