Takagi Yuko, Sindkar Shalaka, Ekonomidis Dimitra, Hall Megan P, Ho C Kiong
Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.
J Biol Chem. 2007 Jun 1;282(22):15995-6005. doi: 10.1074/jbc.M701569200. Epub 2007 Apr 6.
The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.
动质体mRNA的5'端具有高度甲基化的帽4结构，它由标准的m7GpppN（帽0）衍生而来，在剪接前导RNA的前四个核苷内的七个位点有额外的甲基化。除了TbCe1鸟苷酸转移酶和TbCmt1（鸟嘌呤N-7）甲基转移酶外，布氏锥虫还编码第二种帽0形成酶。TbCgm1（布氏锥虫帽鸟苷酸转移酶-甲基转移酶）是一种新型的双功能加帽酶，由一个氨基末端鸟苷酸转移酶结构域和一个羧基末端甲基转移酶结构域组成。重组TbCgm1通过共价酶-GMP中间体将GMP转移到剪接前导RNA（SL RNA）上，并将GpppN末端RNA的鸟嘌呤N-7位甲基化以形成帽0结构。这两个结构域在体外可自主发挥功能。TbCGM1对寄生虫生长至关重要。通过RNA干扰使TbCGM1沉默会增加未加帽的SL RNA的丰度，并导致低甲基化SL RNA的积累。相比之下，TbCE1和TbCMT1的沉默不影响寄生虫生长或SL RNA加帽。我们得出结论，TbCgm1特异性地为SL RNA加帽，帽0是导致成熟SL RNA形成的后续甲基化事件的先决条件。
