Novel upstream and downstream sequence elements contribute to polyadenylation efficiency
Novel upstream and downstream sequence elements contribute to polyadenylation efficiency
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DOI：
10.4161/rna.21957
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发表时间：
2012-10-01
期刊：
影响因子：
4.1
通讯作者：
Lutz, Carol S.
中科院分区：
文献类型：
--
作者：
Darmon, Sarah K.;Lutz, Carol S.
多聚腺苷酸化是3' mRNA加工事件，其通过影响mRNA的稳定性、输出和翻译而有助于基因表达。人多聚腺苷酸化信号（PAS）具有核心和辅助元件，其结合切割位点上游和下游的多聚腺苷酸化因子。大多数mRNA没有最佳的上游和下游核心元件，因此辅助元件可以帮助多聚腺苷酸化效率。我们以前使用了一个全球性的方法来检查辅助元素，以确定过度代表的图案的生物信息学调查。这些预测的信息用于指导我们的体内验证研究，所有这些研究都是使用串联体内聚腺苷酸化测定和使用报告蛋白测定（测量荧光素酶活性）来完成的。将新的辅助元件置于测试多聚腺苷酸化信号中。使用体内聚腺苷酸化测定来确定聚腺苷酸化信号的强度。除了一个新的辅助元件之外，所有的辅助元件都增强了测试聚腺苷酸化信号。当置于萤火虫荧光素酶报告基因的3'UTR中时，还通过荧光素酶测定来测量这些新型辅助元件的作用。两个新型下游辅助元件和所有新型上游辅助元件均显示报告蛋白水平的增加。许多众所周知的辅助多聚腺苷酸化元件已被发现存在于多组中。然而，在我们的研究中，新型辅助元件的多个拷贝使报告蛋白水平以及多聚腺苷酸化选择回到野生型水平。这些新型辅助元件的结构特征也可能影响辅助元件的作用。位于多聚腺苷酸化信号上游的MS2结构可以在正方向和负方向上影响多聚腺苷酸化。通过使用新的互补辅助元件在RNA结构上的大的改变也降低了多聚腺苷酸化选择和报告蛋白水平。因此，我们得出结论，RNA结构在聚腺苷酸化效率中具有重要作用。
Polyadenylation is a 3' mRNA processing event that contributes to gene expression by affecting stability, export and translation of mRNA. Human polyadenylation signals (PAS) have core and auxiliary elements that bind polyadenylation factors upstream and downstream of the cleavage site. The majority of mRNAs do not have optimal upstream and downstream core elements and therefore auxiliary elements can aid in polyadenylation efficiency.Auxiliary elements have previously been identified and studied in a small number of mRNAs. We previously used a global approach to examine auxiliary elements to identify overrepresented motifs by a bioinformatic survey. This predicted information was used to direct our in vivo validation studies, all of which were accomplished using both a tandem in vivo polyadenylation assay and using reporter protein assays measured as luciferase activity. Novel auxiliary elements were placed in a test polyadenylation signal. An in vivo polyadenylation assay was used to determine the strength of the polyadenylation signal. All but one of the novel auxiliary elements enhanced the test polyadenylation signal. Effects of these novel auxiliary elements were also measured by a luciferase assay when placed in the 3' UTR of a firefly luciferase reporter. Two novel downstream auxiliary elements and all of the novel upstream auxiliary elements showed an increase in reporter protein levels.Many well known auxiliary polyadenylation elements have been found to occur in multiple sets. However, in our study, multiple copies of novel auxiliary elements brought reporter protein levels as well as polyadenylation choice back to wild type levels. Structural features of these novel auxiliary elements may also affect the role of auxiliary elements. A MS2 structure placed upstream of the polyadenylation signal can affect polyadenylation in both the positive and negative direction. A large change in RNA structure by using novel complementary auxiliary element also decreased polyadenylation choice and reporter protein levels. Therefore, we conclude that RNA structure has an important role in polyadenylation efficiency.