Pseudogenes are generally considered to be non-functional DNA sequences that arise through nonsense or frame-shift mutations of protein-coding genes1. Although certain pseudogene-derived RNAs have regulatory roles2, and some pseudogene fragments are translated3, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, which reflect low selection pressures on loci no longer relevant to the fitness of a species4. Here we report the characterization of a pseudogene in the chemosensory variant ionotropic glutamate receptor repertoire5,6 of Drosophila sechellia, an insect endemic to the Seychelles that feeds almost exclusively on the ripe fruit of Morinda citrifolia7. This locus, D. sechellia Ir75a, bears a premature termination codon (PTC) that appears to be fixed in the population. However, D. sechellia Ir75a encodes a functional receptor, owing to efficient translational read-through of the PTC. Read-through is detected only in neurons and is independent of the type of termination codon, but depends on the sequence downstream of the PTC. Furthermore, although the intact Drosophila melanogaster Ir75a orthologue detects acetic acid—a chemical cue important for locating fermenting food8,9 found only at trace levels in Morinda fruit10—D. sechellia Ir75a has evolved distinct odour-tuning properties through amino-acid changes in its ligand-binding domain. We identify functional PTC-containing loci within different olfactory receptor repertoires and species, suggesting that such ‘pseudo-pseudogenes’ could represent a widespread phenomenon.
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Salmena, L. Pseudogene redux with new biological significance. Methods Mol. Biol. 1167, 3–13 (2014)
Poliseno, L. et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465, 1033–1038 (2010)
Ji, Z., Song, R., Regev, A. & Struhl, K. Many lncRNAs, 5′UTRs, and pseudogenes are translated and some are likely to express functional proteins. eLife 4, e08890 (2015)
Nei, M., Niimura, Y. & Nozawa, M. The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity. Nat. Rev. Genet. 9, 951–963 (2008)
Benton, R., Vannice, K. S., Gomez-Diaz, C. & Vosshall, L. B. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149–162 (2009)
Croset, V. et al. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet. 6, e1001064 (2010)
Stensmyr, M. C. Drosophila sechellia as a model in chemosensory neuroecology. Ann. NY Acad. Sci. 1170, 468–475 (2009)
Gorter, J. A. et al. The nutritional and hedonic value of food modulate sexual receptivity in Drosophila melanogaster females. Sci. Rep. 6, 19441 (2016)
Becher, P. G., Bengtsson, M., Hansson, B. S. & Witzgall, P. Flying the fly: long-range flight behavior of Drosophila melanogaster to attractive odors. J. Chem. Ecol. 36, 599–607 (2010)
Farine, J. P., Legal, L., Moreteau, B. & Le Quere, J. L. Volatile components of ripe fruits of Morinda citrifolia and their effects on Drosophila. Phytochemistry 41, 433–438 (1996)
Silbering, A. F. et al. Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems. J. Neurosci. 31, 13357–13375 (2011)
Yao, C. A., Ignell, R. & Carlson, J. R. Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. J. Neurosci. 25, 8359–8367 (2005)
Abuin, L. et al. Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69, 44–60 (2011)
Jungreis, I. et al. Evidence of abundant stop codon readthrough in Drosophila and other metazoa. Genome Res. 21, 2096–2113 (2011)
Dunn, J. G., Foo, C. K., Belletier, N. G., Gavis, E. R. & Weissman, J. S. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster. eLife 2, e01179 (2013)
Namy, O. et al. Identification of stop codon readthrough genes in Saccharomyces cerevisiae. Nucleic Acids Res. 31, 2289–2296 (2003)
Legrand, D., Vautrin, D., Lachaise, D. & Cariou, M. L. Microsatellite variation suggests a recent fine-scale population structure of Drosophila sechellia, a species endemic of the Seychelles archipelago. Genetica 139, 909–919 (2011)
Shchedrina, V. A. et al. Analyses of fruit flies that do not express selenoproteins or express the mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance. J. Biol. Chem. 286, 29449–29461 (2011)
Chan, P. P. & Lowe, T. M. GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009)
Palladino, M. J., Keegan, L. P., O’Connell, M. A. & Reenan, R. A. dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing. RNA 6, 1004–1018 (2000)
Irimia, M. et al. A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell 159, 1511–1523 (2014)
Namy, O. & Rousset, J. P. in Recoding: Expansion of Decoding Rules Enriches Gene Expression (eds Atkins, J. F. & Gesteland, R. F. ) 79–100 (Springer, 2010)
Kopczynski, J. B., Raff, A. C. & Bonner, J. J. Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae. Mol. Gen. Genet. 234, 369–378 (1992)
Washburn, T. & O’Tousa, J. E. Nonsense suppression of the major rhodopsin gene of Drosophila. Genetics 130, 585–595 (1992)
Samson, M. L., Lisbin, M. J. & White, K. Two distinct temperature-sensitive alleles at the elav locus of Drosophila are suppressed nonsense mutations of the same tryptophan codon. Genetics 141, 1101–1111 (1995)
Keeling, K. M., Xue, X., Gunn, G. & Bedwell, D. M. Therapeutics based on stop codon readthrough. Annu. Rev. Genomics Hum. Genet. 15, 371–394 (2014)
Jagannathan, S. & Bradley, R. K. Translational plasticity facilitates the accumulation of nonsense genetic variants in the human population. Genome Res. http://dx.doi.org/10.1101/gr.205070.116 (2016)
Huang, W. et al. Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines. Genome Res. 24, 1193–1208 (2014)
Pei, B. et al. The GENCODE pseudogene resource. Genome Biol. 13, R51 (2012)
Adams, M. D. et al. The genome sequence of Drosophila melanogaster. Science 287, 2185–2195 (2000)
Clark, A. G. et al. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450, 203–218 (2007)
Bischof, J., Maeda, R. K., Hediger, M., Karch, F. & Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl Acad. Sci. USA 104, 3312–3317 (2007)
Carracedo, M. C., Asenjo, A. & Casares, P. Genetics of Drosophila simulans male mating discrimination in crosses with D. melanogaster. Heredity 91, 202–207 (2003)
Grosjean, Y. et al. An olfactory receptor for food-derived odours promotes male courtship in Drosophila. Nature 478, 236–240 (2011)
Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999)
Bellen, H. J. et al. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761–781 (2004)
Cook, R. K. et al. The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome. Genome Biol. 13, R21 (2012)
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. & Yamamoto, D. The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124, 761–771 (1997)
Mackay, T. F. et al. The Drosophila melanogaster Genetic Reference Panel. Nature 482, 173–178 (2012)
Grenier, J. K. et al. Global diversity lines—a five-continent reference panel of sequenced Drosophila melanogaster strains. G3 (Bethesda) 5, 593–603 (2015)
Shiao, M. S. et al. Expression divergence of chemosensory genes between Drosophila sechellia and its sibling species and its implications for host shift. Genome Biol. Evol. 7, 2843–2858 (2015)
Arguello, J. R. et al. Extensive local adaptation within the chemosensory system following Drosophila melanogaster’s global expansion. Nat. Commun. 7, ncomms11855 (2016)
Saina, M. & Benton, R. Visualizing olfactory receptor expression and localization in Drosophila. Methods Mol. Biol. 1003, 211–228 (2013)
Benton, R., Vannice, K. S. & Vosshall, L. B. An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature 450, 289–293 (2007)
Benton, R. & Dahanukar, A. Electrophysiological recording from Drosophila olfactory sensilla. Cold Spring Harb. Protoc. 2011, 824–838 (2011)
Kaufman, L. & Rousseeuw, P. J. Finding Groups in Data: an Introduction to Cluster Analysis. (Wiley-Interscience, 2005)
Pei, J., Kim, B. H. & Grishin, N. V. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 36, 2295–2300 (2008)
Jones, D. T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999)
Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)
Armstrong, N. & Gouaux, E. Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28, 165–181 (2000)
We acknowledge C. Carracedo, P. Casares, the Bloomington Drosophila Stock Center (NIH P40OD018537), the Drosophila Species Stock Center (UCSD), and the Developmental Studies Hybridoma Bank (NICHD of the NIH, University of Iowa) for reagents. We thank members of the Benton laboratory for discussions and comments on the manuscript. L.L.P.-G. was supported by a FEBS long-term fellowship; R.R. was supported by a Roche Research Foundation fellowship. J.R.A. was supported by a post-doctoral fellowship from Novartis Foundation for medical–biological research (12A14). M.D.P.’s laboratory was supported by the SNSF. Research in R.B.’s laboratory was supported by ERC Starting Independent Researcher and Consolidator Grants (205202 and 615094), an HFSP Young Investigator Award (RGY0073/2011) and the SNSF Nano-Tera Envirobot project (20NA21_143082).
The authors declare no competing financial interests.
Reviewer Information Nature thanks A. Jacobson, M. Stensmyr and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Quantification of efficiency and tissue-specificity of translational read-through of the D. sechellia Ir75a PTC.
Quantification of GFP staining in the cell bodies of neurons expressing different read-through reporter constructs in different populations of OSNs (see Figs 2, 3 for genotypes). GFP fluorescence levels were normalized by anti-Ir75a fluorescence levels in the Cy3 channel within each analysed cell. Box plots indicate the median and first and third quartile of the data. *P < 0.05, ***P < 0.0005, not significant (n.s.) P > 0.05 (all data analysed using pairwise Wilcoxon rank-sum test, Benjamini–Hochberg correction).
Extended Data Figure 2 Tissue specificity of translational read-through of the D. sechellia Ir75a PTC.
Immunofluorescence with anti-GFP (green) and the neuron nuclear marker anti-Elav (magenta) on whole-mount D. melanogaster antennae in which actin5C-GAL4 drives broad expression of D. sechellia Ir75a*214Q:GFP (UAS-DsIr75a*214Q:GFP/act5C-GAL4) or Ir75a:GFP (UAS-DsIr75a:GFP/act5C-GAL4). Arrowheads indicate examples of GFP-expressing, Elav-negative, non-neuronal cells that were observed in 6 out of 6 antennae expressing the control transgene lacking the PTC, and in 0 out of 6 antennae expressing the PTC-containing transgene. Note that the neuronal GFP signal of both transgenes is heterogeneous across the antenna, possibly because of the variable strength of driver expression and/or instability of the GFP-tagged receptors in heterologous neurons. Scale bars, 10 μm.
Protein-sequence alignment of D. melanogaster, D. simulans and D. sechellia Ir75a. Blue bars indicate the S1 and S2 lobes of the predicted LBD. The position of the PTC (X) is highlighted in yellow. Dark grey columns in the alignment highlight amino acids conserved only in two of the three species. Pink and red shading represents D. sechellia-specific amino acid changes within the LBD; red denotes the subset located in the internal cavity of the binding pocket (Fig. 4a). The locations of the peptide epitopes for the Ir75a antibodies are highlighted with green dashed boxes.
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Prieto-Godino, L., Rytz, R., Bargeton, B. et al. Olfactory receptor pseudo-pseudogenes. Nature 539, 93–97 (2016). https://doi.org/10.1038/nature19824
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