Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans - PubMed (original) (raw)

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

Sarah E Calvo et al. Proc Natl Acad Sci U S A. 2009.

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

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.

Fig. 2.

Protein expression of uORF-containing genes. (A–D) Cumulative distribution of protein expression for mouse genes containing uORFs (red curve) or lacking uORFs (gray curve) in each of 4 independent MS/MS studies (–15). N indicates the number of unique genes in each set. (E) Median reduction of protein and mRNA expression for genes containing uORFs compared with genes lacking uORFs, with P values (in parentheses) computed by empirical permutation testing.

Fig. 3.

Luciferase assays of uORF effects on protein and mRNA levels. (A) Experimental design of reporter constructs with and without uORFs is shown for example Mrpl11. (B–I) Normalized luciferase activity (B–E) and mRNA expression (F–I) are shown for reporter constructs that contain a uORF (red) or lack a uORF (gray) due to a mutation that disrupts the uORF start codon. The constructs contain 5′ UTRs from: 5 mouse genes chosen randomly (B and F), 10 mouse genes with proteomic and conservation signatures of functional uORFs (C and G), 5 human genes with polymorphic uORFs (D and H), and 5 human disease genes with uORF-altering mutations detected in patients (E and I). Error bars represent ±SE of ≥6 biological replicates (B–E) and ≥4 technical replicates (F–I). Asterisks indicate significant difference (P < 0.01).

Fig. 4.

Polymorphic uORF alters FXII protein expression. (A) 5′ UTR sequence of FXII shown with 2 SNP variants, where the T allele creates a uORF (red text). Below are 8 constructs with introduced mutations (underlined text), where colored text indicates a uORF (red) or in-frame alternative start (green). (B) Luciferase activity from reporter constructs listed in A. Error bars represent ±SD of ≥6 biological replicates. (C) Metaanalysis of plasma FXII activity levels measured by 5 independent studies, stratified by genotype of SNP rs1801020.

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Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans - PubMed (original) (raw)