Systematic Dissection of the Sequence Determinants of Gene 3’ End Mediated Expression Control

Abstract

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.

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.…”

Variant Interpretation: Functional Assays to the Rescue

“…14,47,48 MAVEs have also been developed to probe the effects of variants in untranslated regions of mRNAs on message stability and protein expression. [16][17][18] Variation in transcriptional regulatory elements is also important, and pathogenic regulatory variants have been identified for a number of Mendelian disorders. [49][50][51][52] One specific example is the alteration of SORT1 (MIM: 602458) expression by a transcriptional regulatory variant that can increase the risk of myocardial infarction by 40%.…”

“…14,15 The effect of variants on mRNA stability and translation can also be measured by multiplex assays. [16][17][18] Finally, deep mutational scans query the effect of amino acid substitutions on protein function. 19 Because these multiplexed functional assays have the capacity to test 10 4 -10 6 variants per experiment, they are already at the scale required for tackling the VUS problem.…”

Functional genomics bridges the gap between quantitative genetics and molecular biology

“…They can circumvent caveats of linkage disequilibrium obscuring the identity of the causal variant and the bias toward capturing mainly existing common variants. Massively parallel reporter assays allow multiplexed analysis of sequences that control gene expression in vitro, with reporter bar codes that are analyzed by sequencing (Arnold et al 2013;Kheradpour et al 2013;Shalem et al 2015). These assays have provided a wealth of evidence of the function of regulatory elements of the genome, allow precise perturbation of the genetic code, and their high throughput yields comprehensive data for computational analysis of sequence motifs and their function.…”