A systematic study of gene expression variation at single-nucleotide resolution reveals widespread regulatory roles for uAUGs

Abstract

Regulatory single-nucleotide polymorphisms (rSNPs) alter gene expression. Common approaches for identifying rSNPs focus on sequence variants in conserved regions; however, it is unknown what fraction of rSNPs is undetectable using this approach. We present a systematic analysis of gene expression variation at the single-nucleotide level in the Saccharomyces cerevisiae GAL1-10 regulatory region. We exhaustively mutated nearly every base and measured the expression of each variant with a sensitive dual reporter assay. We observed an expression change for 7% (43/582) of the bases in this region, most of which (35/43, 81%) reside in conserved positions. The most dramatic changes were caused by variants that produced AUGs upstream of the translation start (uAUGs), and we sought to understand the consequences and molecular mechanisms underlying this class of mutations. A genome-wide analysis showed that genes with uAUGs display significantly lower mRNA and protein levels than genes without uAUGs. To determine the generality of this mechanism, we introduced uAUGs into S. cerevisiae genes and observed significantly reduced expression in 17/21 instances (p < 0.01), suggesting that uAUGs are functional in a wide variety of sequence contexts. Quantification of mRNA and protein levels for uAUG mutants showed that uAUGs affect both transcription and translation. Expression of uAUG mutants under the upf1D strain demonstrated that uAUGs stimulate the nonsense-mediated decay pathway. Our results suggest that uAUGs are potent and widespread regulators of gene expression that act by attenuating both protein and RNA levels.[Supplemental material is available for this article.]Regulatory single-nucleotide polymorphisms (rSNPs) have garnered much attention in recent biomedical studies. Evidence has revealed that rSNPs contribute to human phenotypic variation and can affect disease susceptibility. Furthermore, many disease-associated SNPs identified in genome-wide association studies (GWAS) are noncoding and are most likely regulatory in nature (Hindorff et al. 2009). However, the identification of rSNPs remains challenging. Many researchers have applied computational methods to distinguish functional rSNPs from a large number of neutral noncoding variations, mostly focusing on SNPs in conserved regions. While such approaches have identified many functional regulatory regions, it is not clear whether they can identify the majority of regulatory elements. For example, a recent study analyzed transcription factor binding sites in five different vertebrates and found that most binding events were species-specific. In fact, for one of their transcription factors, CEPBA, only 0.3% of binding sites were conserved across all five species (Schmidt et al. 2010). Transcription factor binding site (TFBS) ''turnover'' and sequence mutation of binding sites are two mechanisms that may explain this high degree of species-specific binding (Odom et al. 2007;Schmidt et al. 2010). These results raise an important question: How sensitive are alignment-...

Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast

“…Many of these elements were shown to control protein levels by altering the efficiency of translation (7-9, 11), whereas some elements affect translation and in addition, transcription (15) or mRNA degradation (23). Despite much progress, significant challenges remain in deciphering the rules by which multiple elements combine to fine-tune protein expression, as well as in quantifying the degree of expression variation that may be jointly explained by known and novel regulatory elements.Traditionally, the impact of 5′-UTR elements on protein abundance is determined experimentally by comparing a perturbed 5′-UTR sequence with an appropriate control (16,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Although valuable insights emerged from such studies, they frequently focused on the influence of sequence manipulations on a single type of regulatory element.…”

Noise–mean relationship in mutated promoters

“…Values for slopes are provided in Supplemental Table S5, and values for the fold response of each promoter are provided as Supplemental Data. only (e.g., Yun et al 2012), and therefore do not distinguish between mutations affecting burst frequency versus those that modulate burst size. In this study, we relied on the theoretical idea of using gene expression noise to distinguish between those two processes in order to examine the sensitivity of burst size and burst frequency to mutations in promoter sequence.…”

“…In the latter case, the upstream ATG probably reduces burst size via nonproductive translation, although some YFP is generated from the original ATG due to ''slippage'' in translation initiation or reinitiation (Meijer and Thomas 2002;Sachs and Geballe 2006). The upstream ATG may also lower transcript levels (Yun et al 2012). The two types of mutations, TATA and upstream ATGs, also appeared in deviating variants from other promoters (Table 1).…”

“…Still, the principles by which promoter sequences regulate those two processes are not understood, primarily because most studies analyzing the interplay between promoter sequence variations and gene expression consider mean expression only (e.g., Yun et al 2012).…”

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

“…Recently, a systematic study in Saccharomyces cerevisiae showed that uAUGs exert potent and widespread regulation in both transcription and translation levels (97). Mapping of transcript leaders (5=-UTRs that may contain upstream open reading frames) in translation (TL-seq) revealed that uAUGs are rare in yeast, but when present, they are conserved and functional (4).…”