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

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.

基因组3'端区域（3’ end genomic region）编码一系列调控过程，涵盖mRNA稳定性、3'端加工（3’ end processing）与翻译。本研究通过检测13000种设计合成的3'端序列变异体对酵母组成型表达水平的影响，系统探究了3'端介导的表达调控的序列决定因素。本次设计的数据集包含超过200个天然3'端序列的高分辨率扫描诱变变异体，分析结果显示，绝大多数突变仅对表达水平产生微弱影响；且90%左右的强效应突变均定位至单一阳性富TA（TA-rich element）元件——该元件与此前报道的3'端加工效率元件类似——此类突变可导致表达水平最高下降10倍。对3'非翻译区（3' UTR）长度的检测表明，此类突变会使mRNA产生异常延长的3'非翻译区，证实了该元件在3'端加工过程中的作用。有趣的是，本研究发现，此前文献中报道的属于多聚腺苷酸化信号（polyadenylation signal）的其他序列元件，仅对表达水平产生轻微影响。我们进一步通过额外的合成3'端序列表征了该富TA元件的序列特异性，结果显示其活性对单碱基突变敏感，且强烈依赖于侧翼序列的A/T碱基占比。最终，通过计算模型（computational model）分析，我们证实该元件在天然3'端序列中的强度可部分解释其测得的表达变异程度（R = 0.41）。综上，本研究结果强调了高效3'端加工对内源蛋白质水平的重要性，并加深了人们对该过程中涉及的序列元件的理解。