Effects of sequence motifs in the yeast 3\u2032 untranslated region determined from massively parallel assays of random sequences

Andrew Savinov,Josh T Cuperus,Brooke E Angell,Stanley Fields,Benjamin M Brandsen

doi:10.1186/s13059-021-02509-6

Abstract

BackgroundThe 3′ untranslated region (UTR) plays critical roles in determining the level of gene expression through effects on activities such as mRNA stability and translation. Functional elements within this region have largely been identified through analyses of native genes, which contain multiple co-evolved sequence features.ResultsTo explore the effects of 3′ UTR sequence elements outside of native sequence contexts, we analyze hundreds of thousands of random 50-mers inserted into the 3′ UTR of a reporter gene in the yeast Saccharomyces cerevisiae. We determine relative protein expression levels from the fitness of transformants in a growth selection. We find that the consensus 3′ UTR efficiency element significantly boosts expression, independent of sequence context; on the other hand, the consensus positioning element has only a small effect on expression. Some sequence motifs that are binding sites for Puf proteins substantially increase expression in the library, despite these proteins generally being associated with post-transcriptional downregulation of native mRNAs. Our measurements also allow a systematic examination of the effects of point mutations within efficiency element motifs across diverse sequence backgrounds. These mutational scans reveal the relative in vivo importance of individual bases in the efficiency element, which likely reflects their roles in binding the Hrp1 protein involved in cleavage and polyadenylation.ConclusionsThe regulatory effects of some 3′ UTR sequence features, like the efficiency element, are consistent regardless of sequence context. In contrast, the consequences of other 3′ UTR features appear to be strongly dependent on their evolved context within native genes.

Highlights

The 3′ untranslated region (UTR) plays critical roles in determining the level of gene expression through effects on activities such as mRNA stability and translation
To investigate the generalizability and properties of the efficiency element–positioning element interaction, we considered the fraction of N50 sequences containing the canonical consensus forms of both elements (UAUAUA and AAWAAA) in which the efficiency element is 5′ of the positioning element, across 3′ UTRs falling into different enrichment score bins
Some Puf protein binding sites increase protein expression in a random sequence context We examined the results of the N50-C library selection on another class of 3′ UTR sequence elements—Puf protein binding sites—including binding site motifs for Puf1 and Puf2, Puf3, Puf4, Puf5, and Puf6

Summary

Introduction

The 3′ untranslated region (UTR) plays critical roles in determining the level of gene expression through effects on activities such as mRNA stability and translation. Functional elements within this region have largely been identified through analyses of native genes, which contain multiple co-evolved sequence features. Additional facets of gene regulation might be learned by systematically interrogating the functional consequences of libraries of random synthetic sequences whose size vastly exceeds the number of an organism’s genes. Enabled by advances in high-throughput sequencing and oligonucleotide synthesis, this approach has been taken to develop a deeper understanding of 5′ untranslated regions (UTRs) of mRNAs [1, 2], promoters [3, 4], and splicing [5]

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Results

Discussion

Conclusion