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

RNA甲基化作为基因表达调节剂迅速出现。在布氏锥虫及相关寄生虫中，加帽mRNA的5′端存在7种RNA甲基化，可能通过调节蛋白质合成在转录后基因表达中发挥作用。这使得锥虫成为mRNA帽甲基化研究的突出靶点。我们发现，假定的RNA甲基转移酶具有一种新的帽依赖性RNA甲基转移酶活性。重组酶能够将甲基转移到加帽的RNA上。m7 G帽和2′-O核糖甲基化的存在增强了该酶的活性。我们计划通过质谱分析RNA产物来确定这种酶的位置和支持何种RNA修饰，并期望通过在T. cruzi和布氏锥虫。我们构建了在四环素诱导型T7 RNA聚合酶启动子控制下编码Cas9-EGFP蛋白和指导RNA的靶向质粒。稳定的T。以cruzi细胞系为实验对象，分析系统的有效性。作为一种替代和补充的方法，我们还制备了靶向质粒以消除T.布鲁塞。从CRISPR敲除和RNAi敲除细胞系中分离的RNA的比较性全基因组分析应该提供对RNA甲基化如何在真核基因表达中发挥作用的深入了解。

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.