5 prime mRNA cap modifications involved in gene expression control in T. brucei

RNA甲基化正在迅速成为一种基因表达调节因子。在布氏锥虫及其相关寄生虫中，在5‘端存在7个RNA甲基化，可能通过调节蛋白质的合成而影响转录后基因的表达。这使得锥虫成为mRNA帽甲基化研究的突出靶点。我们证明了推测的RNA甲基转移酶具有一种新的帽依赖的RNA甲基转移酶活性。重组酶能将甲基团转移到封端的RNA上。M7G帽和2‘-O核糖甲基化的存在增强了该酶的活性。然而，它似乎没有甲基化已知的甲基化发现的位置。我们计划通过质谱分析RNA产物来确定该酶支持的RNA修饰的位置和类型。我们希望通过在克氏锥虫和布鲁氏锥虫中分别产生CRISPR敲除和RNAi敲除来确定RNA甲基转移酶在寄生虫基因表达中的作用。在四环素可诱导的T7 RNA聚合酶启动子的控制下，我们构建了编码Cas9-EGFP蛋白和引导RNA的靶向载体。获得了一个稳定的克氏毛滴虫细胞系，用于分析该系统的有效性。作为一种替代和补充方法，我们还制备了靶向载体以耗尽布氏毛滴虫的RNA甲基转移酶。对从CRISPR基因敲除和RNAi基因敲除细胞系中分离出来的RNA进行比较的全基因组分析应该有助于深入了解RNA甲基化如何在真核基因表达中发挥作用。

RNA methylation is rapidly emerging as a gene expression regulator. In Trypanosoma brucei and related parasites, seven RNA methylations are present at the 5′-end of capped mRNA, which may play a role in post-transcriptional gene expression by regulating protein synthesis. This makes the Trypanosome an outstanding target for mRNA cap methylation studies.We showed that putative RNA methyltransferase possesses a novel cap-dependent RNA methyltransferase activity. The recombinant enzyme was capable of transferring the methyl group to the capped RNA. The activity was enhanced by the presence of m7G cap and 2′-O ribose methylations. However, it does not appear to methylate the known methylation site found. We plan to determine the position and what kind of RNA modification this enzyme support by analyzing the RNA products using mass spectrometry.We expect to identify the role of the RNA methyltransferase in parasite gene expression by generating CRISPR knock-out and RNAi knockdown in T. cruzi and T.brucei, respectively. We constructed a targeting plasmid encoding for Cas9-EGFP protein and guide RNA under the control of a tetracycline-inducible T7 RNA polymerase promoter. A stable T. cruzi cell line was obtained to analyze the system's effectiveness. As an alternative and complementary approach, we also prepared targeting plasmid to deplete the RNA methyltransferase in T. brucei. Comparative, genome-wide analyses of RNA isolated from CRISPR knock-out and RNAi knockdown cell lines should provide insight into how RNA methylation plays a role in eukaryotic gene expression.