Nprl3 is required for normal development of the cardiovascular system Monika S. Kowalczyk • Jim R. Hughes • Christian Babbs • Luis Sanchez-Pulido • Dorota Szumska • Jacqueline A. Sharpe • Jacqueline A. Sloane-Stanley • Gillian M. Morriss-Kay • Leslie B. Smoot • Amy E. Roberts • Hugh Watkins • Shoumo Bhattacharya • Richard J. Gibbons • Chris P. Ponting • William G. Wood • Douglas R. Higgs Received: 12 January 2012 / Accepted: 19 March 2012 Ó Springer Science+Business Media, LLC 2012 Abstract C16orf35 is a conserved and widely expressed gene lying adjacent to the human a-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, further- more, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid star- vation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturba- tion of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormali- ties and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the car- diovascular system. Introduction Of the *22,000 genes currently annotated in the human genome (GRCh37, 59.37d, Ensembl), the true function is known for only a fraction. Identifying the role played by uncharacterized genes and the proteins they encode is still an important task for fully annotating the functional gen- ome. C16orf35 is a highly conserved gene that lies upstream of the human a-globin cluster in all vertebrates tested (Hughes et al. 2005). Interestingly, this gene con- tains within its introns three highly conserved regulatory elements (R1–R3) that are required to express the a-globin genes at high levels late in erythroid differentiation. Pre- sumably, these embedded regulatory elements have ensured the close linkage (conserved synteny) between C16orf35 and the a-globin genes throughout vertebrate evolution (Hughes et al. 2005). The C16orf35 gene is widely expressed throughout development, predominantly from a housekeeping promoter that in many species is associated with a classical, unmethylated CpG island. However, we have also noted that expression of the Electronic supplementary material The online version of this article (doi:10.1007/s00335-012-9398-y) contains supplementary material, which is available to authorized users. M. S. Kowalczyk Á J. R. Hughes Á C. Babbs Á J. A. Sharpe Á J. A. Sloane-Stanley Á R. J. Gibbons Á W. G. Wood Á D. R. Higgs (&) MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK e-mail: doug.higgs@imm.ox.ac.uk L. Sanchez-Pulido Á C. P. Ponting MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK D. Szumska Á H. Watkins Á S. Bhattacharya Department of Cardiovascular Medicine and Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK G. M. Morriss-Kay Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK L. B. Smoot Á A. E. Roberts Department of Cardiology, Children’s Hospital Boston, Boston, MA 02115, USA 123 Mamm Genome DOI 10.1007/s00335-012-9398-y