5′-UTR序列特征对mRNA丰度的调控作用及优化策略

Regulatory effects of 5′-UTR sequence characteristics on mRNA abundance and optimization strategies

摘要:

目的 转录是基因表达的第一步，mRNA丰度在一定程度上决定着最终蛋白质的表达丰度。近期一些研究发现，5′-UTR中不同的核糖体结合位点(ribosome-binding site, RBS)会对下游基因的mRNA丰度产生影响。从mRNA降解过程的调控因素角度分析，这种影响可能源自Shine-Dalgarno (SD)序列与核糖体的结合强度以及5′-UTR局部二级结构。方法 构建了一个包含518个突变体的5′-UTR突变文库，结合高通量测序技术，高效采集各5′-UTR突变体对应其调控的下游egfp基因的mRNA丰度，并通过RT-qPCR验证了其有效性。结果 将各mRNA突变体丰度与其对应的5′-UTR序列特征进行关联分析，结果表明，与核糖体结合强度为中等偏强的SD序列最有利于维持高mRNA丰度，结合强度过高或过低均会导致mRNA丰度的降低；完全保守的核心SD序列(GGAGG)是保证较高结合强度的关键，其保守性下降将导致mRNA丰度明显下降。当不同序列的5′-UTR中SD序列接近，即与核糖体结合强度相似时，其局部二级结构越不稳定，对应的mRNA丰度越高。结论 本研究解析了5′-UTR的各序列特征对mRNA丰度的调控作用，初步建立了其相互之间的定性模型，为代谢工程及基因线路中调控元件的理性设计提供了重要参考。

Abstract:

Objective Transcription is the first step in gene expression, and the mRNA abundance to a certain extent determines the final protein expression abundance. Recent studies have found that different ribosome-binding sites (RBSs) located in the 5′ untranslated region (5′-UTR) can affect the mRNA abundance of the downstream gene. From the perspective of regulatory factors in the mRNA degradation process, the effect may be attributed to the binding strength between the Shine-Dalgarno (SD) sequence and the ribosome and the local secondary structure of the 5′-UTR.Methods We constructed a 5′-UTR mutant library with a size of 528. High-throughput sequencing was employed to efficiently collect the information on the mRNA abundance of downstream egfp corresponding to various 5′-UTR variants. The effectiveness was verified by RT-qPCR.Results The association between abundance of each mRNA mutant and its corresponding 5′-UTR sequence was analyzed. The results showed that the SD sequence with moderate to strong binding strength to the ribosome was most conducive to maintaining high mRNA abundance. Too high or low binding strength will lead to a reduction in the mRNA abundance. The completely conserved core SD sequence (GGAGG) was the key to ensuring high binding strength, and the decline in conservation would cause a significant decrease in the mRNA abundance. When the SD sequence was similar among different 5′-UTR variants, i.e., the binding strength of the SD sequence to the ribosome was comparable, the local secondary structure of the 5′-UTR was instable and the abundance of corresponding mRNA was high.Conclusion This study delves into the regulatory effects of 5′-UTR sequence features 5′-UTR on the mRNA abundance and establishes a qualitative model of their interrelationships, providing a reference for the rational design of regulatory elements in metabolic engineering and gene circuits.