5PSeq supporting study Probing 3’UTRs as modular regulators of gene expression. 5PSeq supporting study Probing 3’UTRs as modular regulators of gene expression

The goal of the study is to analyse the function of 3'-UTR motifs in the non-native context. Several 3'-UTR motifs predicted to have stabilizing or destabilizing effects on mRNA were inserted into terminators controlling the expression of reporter mCherry gene in following contexts: pRps3-mCherry-tRps3, pPgk1-mCherry-tRps3, pTsa1-mCherry-tTsa. 5'P sequencing was used to determine whether or not modifications in the 3'-UTR region affect ribosomal dynamics and co-translational degradation of mRNA in the 3' end region of reporter coding sequence. Overall design: 5'P sequencing of Saccharomyces cerevisiae strains expressing mCherry reporter controlled by modified and non-modified terminators. mCherry reporter constructs are expressed from a low copy number plasmid. All modified constructs have three 9bp insertion sites with different combinations of the following motifs: N, a randomly generated sequence; A, an ATATTC motif; H, a TTTCATTTC motif; and T, a TGTACAATA motif.

本研究旨在探究非天然环境下3'-非翻译区（3'-UTR）基序的功能。已有多个被预测可对mRNA产生稳定或去稳定作用的3'-非翻译区基序，被插入至终止子中，以在以下环境中调控报告基因mCherry的表达：pRps3-mCherry-tRps3、pPgk1-mCherry-tRps3、pTsa1-mCherry-tTsa。本研究采用5'P测序（5'P sequencing）技术，以检测3'-非翻译区的修饰是否会影响核糖体动力学，以及报告基因编码区3'端区域的mRNA共翻译降解过程。
实验整体设计：对携带经修饰与未修饰终止子调控的mCherry报告基因的酿酒酵母（Saccharomyces cerevisiae）菌株开展5'P测序。mCherry报告基因构建体由低拷贝质粒进行表达。所有经修饰的构建体均带有3个9bp插入位点，并搭载以下基序的不同组合：N为随机生成的序列；A为ATATTC基序；H为TTTCATTTC基序；T为TGTACAATA基序。