Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans

Upstream ORFs (uORFs) are mRNA elements defined by a start codon in the 5′ UTR that is out-of-frame with the main coding sequence. Although uORFs are present in approximately half of human and mouse transcripts, no study has investigated their global impact on protein expression. Here, we report that uORFs correlate with significantly reduced protein expression of the downstream ORF, based on analysis of 11,649 matched mRNA and protein measurements from 4 published mammalian studies. Using reporter constructs to test 25 selected uORFs, we estimate that uORFs typically reduce protein expression by 30–80%, with a modest impact on mRNA levels. We additionally identify polymorphisms that alter uORF presence in 509 human genes. Finally, we report that 5 uORF-altering mutations, detected within genes previously linked to human diseases, dramatically silence expression of the downstream protein. Together, our results suggest that uORFs influence the protein expression of thousands of mammalian genes and that variation in these elements can influence human phenotype and disease.

[1]  L. Rubbia‐Brandt,et al.  Severe hemochromatosis in a Portuguese family associated with a new mutation in the 5'-UTR of the HAMP gene. , 2004, Blood.

[2]  David Hogg,et al.  Mutation of the CDKN2A 5' UTR creates an aberrant initiation codon and predisposes to melanoma , 1999, Nature Genetics.

[3]  Daniel J. Blankenberg,et al.  28-way vertebrate alignment and conservation track in the UCSC Genome Browser. , 2007, Genome research.

[4]  Elizabeth M. Smigielski,et al.  dbSNP: the NCBI database of genetic variation , 2001, Nucleic Acids Res..

[5]  S. Carr,et al.  A Mitochondrial Protein Compendium Elucidates Complex I Disease Biology , 2008, Cell.

[6]  Yang Liu,et al.  Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis , 2009, Nature Genetics.

[7]  M. Kozak,et al.  Constraints on reinitiation of translation in mammals. , 2001, Nucleic acids research.

[8]  M. Kozak,et al.  Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. , 1987, Molecular and cellular biology.

[9]  P. K. Chakraborty,et al.  Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype , 2002, Human mutation.

[10]  Matthias Mann,et al.  In-depth Analysis of the Adipocyte Proteome by Mass Spectrometry and Bioinformatics*S , 2007, Molecular & Cellular Proteomics.

[11]  J. McCarthy,et al.  Regulation of fungal gene expression via short open reading frames in the mRNA 5′untranslated region , 2003, Molecular microbiology.

[12]  J. Galagan,et al.  Dual modes of natural selection on upstream open reading frames. , 2007, Molecular biology and evolution.

[13]  Nicholas T. Ingolia,et al.  Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling , 2009, Science.

[14]  J. Fontcuberta,et al.  Association after linkage analysis indicates that homozygosity for the 46C→T polymorphism in the F12 gene is a genetic risk factor for venous thrombosis , 2004, Thrombosis and Haemostasis.

[15]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[16]  B. Boehm,et al.  Coagulation factor XII (FXII) activity, activated FXII, distribution of FXII C46T gene polymorphism and coronary risk , 2008, Journal of thrombosis and haemostasis : JTH.

[17]  Deepak Kolippakkam,et al.  Comprehensive and quantitative proteome profiling of the mouse liver and plasma , 2008, Hepatology.

[18]  F. Rosendaal,et al.  The 46C→T polymorphism in the factor XII gene (F12) and the risk of venous thrombosis , 2005, Journal of thrombosis and haemostasis : JTH.

[19]  S. Tuffery,et al.  Mutation in the 5′ noncoding region of the SRY gene in an XY sex‐reversed patient , 1998, Human mutation.

[20]  Crispin Miller,et al.  Quantitative Proteomics Analysis Demonstrates Post-transcriptional Regulation of Embryonic Stem Cell Differentiation to Hematopoiesis*S , 2008, Molecular & Cellular Proteomics.

[21]  M. Tomita,et al.  Bioinformatic analysis of post‐transcriptional regulation by uORF in human and mouse , 2007, FEBS letters.

[22]  N. Olivieri,et al.  Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene. , 1992, Blood.

[23]  Olfert Landt,et al.  Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis , 2000, Nature Genetics.

[24]  Michelle S. Scott,et al.  Global Survey of Organ and Organelle Protein Expression in Mouse: Combined Proteomic and Transcriptomic Profiling , 2006, Cell.

[25]  Igor Jurisica,et al.  Integrated proteomic and transcriptomic profiling of mouse lung development and Nmyc target genes , 2007, Molecular systems biology.

[26]  P. Stenson,et al.  Human Gene Mutation Database (HGMD®): 2003 update , 2003, Human mutation.

[27]  H. Huopio,et al.  Acute insulin response tests for the differential diagnosis of congenital hyperinsulinism. , 2002, The Journal of clinical endocrinology and metabolism.

[28]  N A Kolchanov,et al.  Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features , 1998, FEBS letters.

[29]  T. Huisman,et al.  The G----A mutation at position +22 3' to the Cap site of the beta-globin gene as a possible cause for a beta-thalassemia. , 1991, Hemoglobin.

[30]  N. Bresolin,et al.  Genetic polymorphisms for the study of multifactorial stroke , 2008, Human mutation.

[31]  H. Meijer,et al.  Control of eukaryotic protein synthesis by upstream open reading frames in the 5'-untranslated region of an mRNA. , 2002, The Biochemical journal.

[32]  S. Batalov,et al.  A gene atlas of the mouse and human protein-encoding transcriptomes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  S. Humphries,et al.  The molecular basis of familial hypercholesterolaemia in Turkish patients. , 2005, Atherosclerosis.

[34]  Y. Agid,et al.  Levodopa-responsive dystonia. GTP cyclohydrolase I or parkin mutations? , 2000, Brain : a journal of neurology.

[35]  N. Hamasaki,et al.  A common genetic polymorphism (46 C to T substitution) in the 5'-untranslated region of the coagulation factor XII gene is associated with low translation efficiency and decrease in plasma factor XII level. , 1998, Blood.

[36]  M. Kozak,et al.  An analysis of vertebrate mRNA sequences: intimations of translational control , 1991, The Journal of cell biology.

[37]  Z. Navrátilová.  Polymorphisms in CCL2&CCL5 chemokines/chemokine receptors genes and their association with diseases. , 2006, Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia.

[38]  Jeffrey C. Murray,et al.  Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes , 2002, Nature Genetics.

[39]  O. Wagner,et al.  A common C-->T polymorphism at nt 46 in the promoter region of coagulation factor XII is associated with decreased factor XII activity. , 2001, Thrombosis research.

[40]  Graziano Pesole,et al.  uAUG and uORFs in human and rodent 5'untranslated mRNAs. , 2005, Gene.

[41]  A. Grüters,et al.  Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans , 1998, Nature Genetics.

[42]  P. Stenson,et al.  Human Gene Mutation Database (HGMD , 2003 .

[43]  Terrence S. Furey,et al.  The UCSC Genome Browser Database , 2003, Nucleic Acids Res..

[44]  R. Skoda,et al.  An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia , 1998, Nature Genetics.

[45]  Steven D Shapiro,et al.  Expression profiling of the developing mouse lung: insights into the establishment of the extracellular matrix. , 2002, American journal of respiratory cell and molecular biology.

[46]  M. Kozak.  Structural features in eukaryotic mRNAs that modulate the initiation of translation. , 1991, The Journal of biological chemistry.

[47]  D. Morris,et al.  Upstream Open Reading Frames as Regulators of mRNA Translation , 2000, Molecular and Cellular Biology.

[48]  T. Huisman,et al.  The G→A Mutation at Position +22 31 to the Cap Site of the β-Globin Gene as a Possible Cause for a β-Thalassemia , 1991 .