A new critical site for spectral tuning of red/green-sensitive visual pigment identified in a murid rodent

With the appearance of eutherian mammals, 3 visual pigments were retained in their retina, mediating light sensitivity. Of them, rhodopsin is expressed in rod photoreceptor cells to conduct dim-light vision, while the other 2 visual pigments, middle/long wavelength-sensitive (M/LWS) and short wavelength-sensitive type 1 (SWS1) visual pigments, expressed in cones are for color vision (Chi et al. 2020). Murid rodents (order Rodentia: family Muridae), originated 17–22 million years ago (Mya) and represent more than 10% of living mammal species (Aghová et al. 2018). Within these murid species, their SWS1 pigment retained the ancestral ultraviolet (UV) sensitivity, which is generally associated with a nocturnal lifestyle (Emerling et al. 2015). In contrast, the spectral sensitivities of murid M/LWS are more diversified, with wavelengths of maximum absorption (λmax) ranging from 490 to 509 nm (Yokoyama et al. 2008; Chi et al. 2020). The spectral tuning of this pigment can generally be predicted from 5 critical sites (180, 197, 277, 285, and 308) (Yokoyama et al. 2008), with 5 additional sites (48, 134, 194, 213, and 294) that could lead to ≥5 nm shift being identified (Matsumoto et al. 2014; Chi et al. 2020; Fujimoto et al. 2023). Interestingly, while showing an identical repertoire for the critical sites of M/LWS (residues at the 10 above sites: ASAYYYYTHS) with the mouse (508 nm) and the rat (509 nm) (Yokoyama et al. 2008), the spectral sensitivity of the striped mouse (Rhabdomys pumilio) is reported to be 500 nm by electroretinogram (ERG) measurement (Allen et al. 2020), suggesting one or more additional unknown critical sites in this species. Thus, in this study, we carried out site-directed mutagenesis and functional assays for the M/LWS pigment of the striped mouse to elucidate the molecular mechanism underlying this color sensitivity shift. To explore the molecular mechanism of M/LWS in the striped mouse, the gene coding sequences from 22 species, including the striped mouse (R. pumilio) and its close relative R. dilectus, from the subfamily Murinae were obtained from the NCBI (www.ncbi.nlm.nih.gov) database for comparison. In addition, M/LWS gene sequences from 2 species from the subfamilies Gerbillinae and Lophiomyinae were used as outgroups (Supplementary Table S1 and Supplementary Materials). After ancestral sequence reconstruction, none of the 10 known critical sites (48 [9 nm], 134 [6 nm], 180 [5 nm], 194 [7 nm], 197 [24 nm], 213 [6 nm], 277 [10 nm], 285 [17 nm], 294 [8 nm], and 308 [33 nm]) (Yokoyama et al. 2008; Matsumoto et al. 2014; Chi et al. 2020; Fujimoto et al. 2023) reveal any amino acid substations in the striped mouse species (R. pumilio) or its close relative (R. dilectus), both of which share identical amino acid sequences for M/LWS (Figure 1A). Spectral tuning of M/LWS pigments in the striped mouse (Rhabdomys pumilio) and the shrew-rat (Rhynchomys soricoides). (A) Molecular evolution of M/LWS in the species tree of the subfamily Murinae (see Supplementary Materials). Amino acid substitutions are shown in the branches, with reported critical sites highlighted in red. Predicted λmax values based on the known critical sites (48, 134, 180, 194, 197, 213, 277, 285, 294, and 308) for living species are shown in italic, where the λmax values of M/LWS experimentally measured for mouse and rat are shown in green (Yokoyama et al. 2008; Matsumoto et al. 2014; Chi et al. 2020; Fujimoto et al. 2023). New experimental data measured from this study (*) for the striped mouse and the shrew-rat are exhibited in blue (497 nm) and green (510 nm), respectively. The amino acid sequences of M/LWS from Rhabdomys pumilio and R. dilectus are identical and thus the λmax values of M/LWS pigments from both striped mice are blue-shifted compared with other species in the tree. Both dark and dark minus light difference spectra (inset) are shown for the 2 rodent species. (B) Sequence alignment of M/LWS between striped mouse and shrew-rat. Aligned site positions are according to the human MWS gene (Accession No. AH005296.2). Sites highlighted with red backgrounds are the 10 known critical sites, and those with red boxes are potentially important positions affecting spectral tuning in this pigment given that they are changes at hydroxyl-bearing residue. To validate the potential short wavelength-shifted phenotype of the M/LWS of this species, in vitro functional assays on this pigment were performed from both the striped mouse (R. pumilio) and the shrew-rat (Rhynchomys soricoides), which is a basal lineage for this clade (Supplementary Materials). Our results show that the M/LWS pigment from the shrew-rat is functionally conserved compared with the mouse and rat, with a λmax at 510 nm (Figure 1A,B, and Supplementary Data S1). In contrast, the pigment from the striped mouse is sensitive at 497 nm, which further supports the color vision of this species is shorter wavelength-shifted as proposed by the ERG experiment (500 nm) (Allen et al. 2020). Thus, there is a 13 nm difference between these 2 murid species, both of which possess identical residues at the 10 known critical sites (Figure 1A,B, and Supplementary Data S1). The blue-shifted sensitivity of M/LWS in the 2 striped mice is parallel to that reported for this pigment from another murid species, the Mongolian gerbil (Meriones unguiculatus), with both groups showing a diel activity transition from nocturnal to diurnal (Chi et al. 2020; Richardson et al. 2023). However, more evidence from behavioral and physiological studies would be helpful to uncover the exact ecological reasons for these color vision shifts. To reveal the amino acid substitution(s) important for this difference between prediction and experimental verification, mutagenesis experiments were carried out in the M/LWS pigment of the striped mouse. Given the reported critical amino acid substitutions are related to hydroxyl-bearing residues (Yokoyama et al. 2008), single amino acid mutants were generated by PCR at 6 positions (11, 16, 25, 57, 58, and 110) individually that differ between the striped mouse and shrew-rat and involve a substitution that changes a hydroxyl-bearing amino acid residue (Figure 1C) (Supplementary Materials). No spectral sensitivity shift was found for the mutants T11P, S16R, S25A, T57S, or A58T (Supplementary Figure S1 and Supplementary Data S1), however, a big λmax-shift (12 nm) was seen at site 110 when it was changed from a cysteine (C) to a serine (S) (Figure 2A and Supplementary Data S1). Both striped mice species (R. pumilio and R. dilectus) share the same codon (TGT) for serine at this site (Figure 2B), further suggesting that this substitution was fixed in their common ancestor and that both of them possess blue-shifted M/LWS (Figure 1A). To further verify the impact of this substitution on spectral tuning, the reverse mutant S110C was generated with the M/LWS pigment of the shrew-rat, a close relative, and again, a 14 nm difference was observed compared with the wild-type pigment (Figure 2A and Supplementary Data S1). Taken together, similar to the other four reported critical sites (197, 277, 285, and 308) (Yokoyama et al. 2008), an amino acid substitution at site 110 could also lead to a >10 nm λmax-shift. Molecular mechanism underlying spectral sensitivity shift in the striped mouse. (A) Spectral tuning of single-mutant C110S on the M/LWS pigment from the striped mouse. A 12 nm λmax-shift toward a longer wavelength-sensitive was observed compared with the wild-type pigment (497 nm, gray dotted line). The reverse single-mutant S110C on the shrew-rat pigment shows a reverse λmax-shift of a 14 nm difference between the mutant and the wild-type pigment (510 nm, gray dotted line). (B) Codon of site 110 across species in the subfamily Murinae. The non-synonymous substitution in the 2 striped mice (black box) is shown in red background. (C) Schematic M/LWS structure with the 11 critical sites shown as large red dots. The newly identified 110 site is highlighted by a “*.” Little blue dots are residues of the transmembrane domains I–VII (from left to right). Site 110 is located in the second transmembrane domain of the M/LWS pigment (Figure 2C) and corresponds to site 94 in bovine rhodopsin (Yamaguchi et al. 2023). Notably, amino acid substitutions at site 94 in both RH1 and SWS2 (short wavelength-sensitive type 2) pigments have been shown impact on λmax-shift (>10 nm) (Patel et al. 2020; Yamaguchi et al. 2023). Thus, our mutagenesis results not only expand the number of critical sites for spectral tuning of M/LWS but also indicates site 110 has a general role in vertebrate visual pigments. This role in visual tuning could be due to its close distance to the chromophore binding pocket where it may affect the hydrogen-bonding network around the Schiff base through polarity changes of residues (Patel et al. 2020; Yamaguchi et al. 2023). This new critical site found in the M/LWS of striped mouse together with the recently reported important ones in another murid rodent (Chi et al. 2020) suggests that more functional studies, and also structural analyses, of this pigment in this species-rich group are necessary for elucidating its mechanisms of spectral tuning. Supplementary material can be found at https://academic.oup.com/cz. We thank Lisa Neuhold from the National Eye Institute (NIH) for providing 11-cis-retinal, and also with the help from Cheryl England and Clint Makino from Boston University. This project was supported by the Natural Science Basic Research Program of Shaanxi (2022JQ-169) and the China Postdoctoral Science Foundation (2021M702063) to S.S., the National Natural Science Foundation of China (32270462), and the Fundamental Research Funds for the Central Universities (GK202401001) to Y.L. The authors have no competing interests. Y.L. conceived the study. Y.L. and S.S. contributed experimental reagents and materials. S.S. and J.Z. performed experiments. S.S., Y.X., and Y.L. analyzed data. S.S., Y.L., and D.M.I. wrote the paper. Ethical approval was not required for the in vitro assays carried out in this study.

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