Monday, April 6, 2015

Everything's comin up peropsin

(Or maybe coming up RGR). My research activities on opsin have lately led me to peropsin and RGR. These are opsins in the RGR/Go superfamily (a la Plachetzki et al 2007) [Note Porter et al 2012 named this superfamily Group IV, but I don't like that name as much because it is not clear there are actually four superfamily groups].

Below is a report I sent to the first author of a paper that just came out. The paper is here:
Battelle, B., Kempler, K., Saraf, S., Marten, C., Dugger, D., Speiser, D., & Oakley, T. (2014). Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes Journal of Experimental Biology, 218 (3), 466-479 DOI: 10.1242/jeb.116087

I've taken to writing reports for collaborations, as it focuses me to get done the work, and to share it with collaborators in a way where they can easily extract information for the publication. Below is such a report that I created for the above-cited paper:

Limulus Peropsin-like gene phylogeny report

In 1997, (Sun et al. 1997) reported a new opsin, found in cDNA libraries of human eyes, and shown by immunohistochemistry to be expressed in Retinal Pigment Epithelium. Another name for peropsin is RRH (retinal pigment epithelium-derived rhodopsin homologue). The most closely related gene to peropsin in the human genome is RGR (RPE−retinal G protein−coupled receptor), first discovered in 1993 (Jiang, Pandey, and Fong 1993). Based on mouse knockout experiments, RGR is a photoisomerase involved in the generation of 11-cis-retinal (Chen et al. 2001). Both of these vertebrate genes belong to a large clade of opsins called “RGR/Go” (Plachetzki, Degnan, and Oakley 2007; Feuda et al. 2012) or “Group-IV” opsins (Porter et al. 2012).

Nagata et al (2010) claimed to find the first peropsin from a protostome, a jumping spider, Hasarius adansoni. However, their phylogenetic analysis showed only weak support (77%) for the spider gene as the sister to peropsins and was based on overly simplistic phylogenetic techniques (neighbor-joining based on an unspecified distance model). Eriksson et al (2013) discovered a gene from the spider Cupiennius salei that is very similar to the jumping spider gene. While their phylogenetic analysis shows good support for these spider genes in the Group IV clade (1.0 posterior probability in Bayesian Inference), their placement with peropsin is again tenuous (0.62). Hering and Mayer (2014) reported a third chelicerate peropsin-like gene from the genome of the spider mite Tetranychus urticae. The three chelicerate genes form a well-supported clade within Group IV opsins, but their phylogenetic position was again uncertain with respect to peropsin and RGR. In some analysis of Hering and Mayer (2014), the chelicerate genes are weakly supported as sister to all other RGR and peropsin genes and in one analysis they are weakly supported as sister to RGR.

I conducted phylogenetic analyses on 30 opsin sequences, including our putative Limulus peropsin/RGR-like gene, 27 genes of the RGR/peropsin (=RPE/peropsin) clade of Hering and Mayer (2014), plus two outgroup opsins with solved crystal structures (Bos taurus c-opsin (Palczewski et al. 2000) and Todarodes r-opsin (Murakami and Kouyama 2008)). I conducted all phylogenetic analyses using the Osiris phylogenetics package (Oakley et al. 2014) within Galaxy (Blankenberg et al. 2005). I first aligned all 30 sequences using MUSCLE (Edgar 2004). I next used RAxML version 7.4 (Stamatakis 2006), assuming a GTR+G model to search for the Maximum Likelihood phylogeny, and conducted 100 bootstrap pseudoreplications to gauge node stability.


The new Limulus peropsin/RGR-like gene forms a clade with the three “peropsins” previously described from chelicerates (100%), and is sister to the two spider “peropsins” (93%). The relationships within chelicerates are not consistent with taxonomy, which would predict that Limulus, as the only non-arachnid, should fall as the sister gene to the other three chelicerate genes. Our results are similar to previous analyses that cannot confidently place the chelicerate genes in a specific position between RGR and peropsin.

We found a gene in Limulus that is very similar to other chelicerate genes known in the literature as protostome “peropsins”. However, a careful examination of previous studes, and our own results, indicate that the chelicerate genes may orthologs of peropsins or RGR genes, or that vertebrate RGR and peropsins are in paralogs compared to the chelicerate genes. The inability of phylogenetic analyses to unequivocally place the chelicerate genes could be caused by sparse sampling of invertebrate genomes. In addition, many of the peropsin/RGR sequences found in invertebrate full genome sequences have not been experimentally verified, nor has function been demonstrated for them. Clearly, there is much to learn about this clade of opsins.