Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans Sarah E Calvoa b c d 1 David J Pagliarinia b c 1 and Vamsi K Moothaa b c 2 aBroad Institute of MIT and Harvard Cambridge MA 02142 bCenter for Human Genetic Research Massachusetts General Hospital Boston MA 02114 of Systems Biology Harvard Medical School Boston MA 02115 and dDivision of Health Sciences and Technology Harvard MIT Cambridge MA 02139 cDepartment Edited by Jonathan Weissman University of California San Francisco CA and accepted by the Editorial Board March 18 2009 received for review October 29 2008 polymorphism post transcriptional control proteomics translation uORF T he regulation of gene expression is controlled at many levels including transcription mRNA processing protein translation and protein turnover Posttranscriptional regulation is often controlled by short sequence elements in the UTRs of mRNA One such 5 UTR element is the upstream ORF uORF depicted in Fig 1A Because eukaryotic ribosomes usually load on the 5 cap of mRNA transcripts and scan for the presence of the first AUG start codon uORFs can disrupt the efficient translation of the downstream coding sequence 1 2 Previous reports have shown that ribosomes encountering a uORF can i translate the uORF and stall triggering mRNA decay ii translate the uORF and then with some probability reinitiate to translate the downstream ORF or iii simply scan through the uORF 2 uORFs have been shown to reduce protein levels in 100 eukaryotic genes supporting information SI Table S1 Additionally mutations that introduce or disrupt a uORF have found to cause 3 human diseases 3 5 In several interesting cases the uORF derived protein is functional however in most cases the mere presence of the uORF is sufficient to reduce expression of the downstream ORF 1 2 6 8 Previous genomic analyses suggest that uORFs may be widely functional for several reasons They correlate with lower mRNA expression levels 9 they are less common in 5 UTRs than would be expected by chance 6 10 they are more conserved than expected when present 6 and several hundred have evidence of translation in yeast 11 However no study has demonstrated that these elements have a widespread impact on cellular protein levels Moreover no study has investigated whether uORF presence varies in the human population Here we take advantage of recently available datasets of protein abundance 12 17 and genetic variation 18 19 to assess the impact and natural variation of mammalian uORFs www pnas org cgi doi 10 1073 pnas 0810916106 A cap main coding sequence 5 UTR AUG AUG uORF uORF 3 UTR AUG B Transcripts with annotated 5 U TR 1 uO RF 2 uO RFs 1 uO RF fully up stream 1 uO RF overlap p ing C D S Median Length nt 5 U TR uO RF polyA AAAAAA Human 23775 11670 6268 9879 4275 Mouse 18663 8253 4197 6935 2872 170 48 139 48 Fig 1 uORF definition and prevalence A Schematic representation of mRNA transcript with 2 uORFs red arrows 1 fully upstream and 1 overlapping the main coding sequence black arrow uORFs are defined by a start codon AUG in the 5 UTR an in frame stop codon arrowhead preceding the end of the main coding sequence and length 9 nt B Number and length of uORFs in human and mouse RefSeq transcripts Results uORF Prevalence Within Mammalian Transcripts We define a uORF as formed by a start codon within a 5 UTR an in frame stop codon preceding the end of the main coding sequence CDS and length at least 9 nt including the stop codon As shown in Fig 1 A this definition includes uORFs both fully upstream and overlapping the CDS because both types are predicted to be functional 20 We searched for uORFs within all human and mouse RefSeq transcripts with annotated 5 UTRs 10 nt Consistent with previous estimates 9 10 we find that 49 of human and 44 of mouse transcripts contain at least 1 uORF Fig 1B Interestingly human and mouse uORF start codons uAUGs are the most conserved 5 UTR trinucleotide across vertebrate species Fig S1 consistent with a widespread functional role uORF Impact on Cellular Protein Levels If uORFs cause widespread reduction in protein expression as predicted by ribosome scanning Author contributions S E C D J P and V K M designed research S E C and D J P performed research and S E C wrote the paper The authors declare no conflict of interest This article is a PNAS Direct Submission J W is a guest editor invited by the Editorial Board Freely available online through the PNAS open access option 1S E C and D J P contributed equally to this work 2To whom correspondence should be addressed at Center for Human Genetic Research Massachusetts General Hospital 185 Cambridge Street CPZN 5 806 Boston MA 02114 E mail vamsi hms harvard edu This article contains supporting information online at www pnas org cgi content full 0810916106 DCSupplemental PNAS May 5 2009 vol 106 no 18 7507 7512 GENETICS 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 7508 www pnas org cgi doi 10 1073 pnas 0810916106 1 0 Fraction of genes A 0 8 liver Lai et al 0 6 0 4 0 2 uORF N 1041 no uORF N 1443 0 0 10 B Fraction of genes 1 0 0 8 100 protein expression 1000 lung development Cox et al 0 6 0 4 0 2 uORF N 201 no uORF N 521 0 0 10 100 protein expression C Fraction of genes 1 0 0 8 D 1000 mitochondria 14 tissues Pagliarini et al 0 6 0 4 uORF N 141 no uORF N 346 0 2 0 0 1 0 Fraction of genes models we would expect uORF containing transcripts to correlate with lower protein levels when