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

Abstract

The 5′-untranslated region (5′-UTR) of mRNAs contains elements that affect expression, yet the rules by which these regions exert their effect are poorly understood. Here, we studied the impact of 5′-UTR sequences on protein levels in yeast, by constructing a large-scale library of mutants that differ only in the 10 bp preceding the translational start site of a fluorescent reporter. Using a high-throughput sequencing strategy, we obtained highly accurate measurements of protein abundance for over 2,000 unique sequence variants. The resulting pool spanned an approximately sevenfold range of protein levels, demonstrating the powerful consequences of sequence manipulations of even 1-10 nucleotides immediately upstream of the start codon. We devised computational models that predicted over 70% of the measured expression variability in held-out sequence variants. Notably, a combined model of the most prominent features successfully explained protein abundance in an additional, independently constructed library, whose nucleotide composition differed greatly from the library used to parameterize the model. Our analysis reveals the dominant contribution of the start codon context at positions −3 to −1, mRNA secondary structure, and out-of-frame upstream AUGs (uAUGs) to phenotypic diversity, thereby advancing our understanding of how protein levels are modulated by 5′-UTR sequences, and paving the way toward predictably tuning protein expression through manipulations of 5′-UTRs.post-transcriptional regulation | computational prediction | AUG sequence context | mRNA folding | upstream start codons T he control of protein expression is a fundamental process of living cells. Much progress has recently been made in understanding how information encoded within DNA regulatory sequences is converted to yield a precise expression output (1-4). However, functional elements are also embedded within RNA sequences, such as 5′-untranslated regions (5′-UTRs). Accumulating evidence has revealed the importance of 5′-UTRs in shaping eukaryotic protein expression (5-12). Such 5′-UTR sequences encode a variety of cis-regulatory elements, including a 5′-cap structure (13), a translation initiation motif (14-16), upstream AUGs (uAUGs) and upstream ORFs (17, 18), internal ribosome entry sites (19), terminal oligo-pyrimidine tracts (20), secondary structures (21), and G-quadruplexes (22). 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)(2...

Cis-regulatory elements explain most of the mRNA stability variation across genes in yeast

“…An alternative approach to the modeling of endogenous sequence is to use large-scale synthetic libraries (Dvir et al 2013;Shalem et al 2015;Wissink et al 2016). Although very powerful to dissect known cis-regulatory elements or to investigate small variations around select genes, the sequence space is so large that these large-scale perturbation screens cannot uncover all regulatory motifs.…”

“…One of the interesting sequence elements that we found associated with half-life but did not turn out significant in the joint model is the start codon context. Given its established effect on translation initiation (Kozak 1986;Dvir et al 2013), the general coupling between translation and mRNA degradation (Roy and Jacobson 2013;Huch and Nissan 2014;, as well as several observations directly on mRNA stability for single genes (LaGrandeur and Parker 1999;Schwartz and Parker 1999), the start codon context may nonetheless functionally affect mRNA stability. Consistent with this hypothesis, large-scale experiments that perturb 5 ′ sequence secondary structure and start codon context indeed showed a wide range of mRNA level changes in the direction that we would predict (Dvir et al 2013).…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Likewise, in studies of start codon context in derivatives of the CYC1 gene, Baim and Sherman (1988) found that U or C at the 23 position resulted in roughly a twofold increase in leaky scanning as compared to when a purine was at this position. Finally, based on a high throughput screen of start codon context nucleotides, Dvir et al (2013) reported a significant, but modest (,30%), impact of a 23 purine on reporter gene expression. At odds with these studies, flanking nucleotides have been shown to be important in selection of the non-AUG start codon on the GRS1 mRNA (Chen et al 2008) as well as in the selection of alternate AUG start codons on the MOD5 and CCA1 mRNAs (Werner et al 1987;Slusher et al 1991;Wolfe et al 1994).…”

The role of RNA structure at 5′ untranslated region in microRNA-mediated gene regulation

“…In contrast, in yeast, the secondary structure of the 5 ′ UTR close to the start codon is selectively loose (Ringnér and Krogh 2005). Mutagenesis studies in both yeast (Dvir et al 2013) and Arabidopsis thaliana (Kim et al 2014) demonstrated that the local RNA secondary structure immediately upstream of the start codon plays a crucial role in determining translational efficiency and thus affects protein production.…”