A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei
A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei
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DOI：
10.1261/rna.2223406
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发表时间：
2006-01-01
期刊：
影响因子：
4.5
通讯作者：
Tschudi, C
中科院分区：
文献类型：
--
作者：
Arhin, GK;Li, HJ;Tschudi, C
剪接前导（SL）RNA通过将m（7）G加帽的SL序列提供给每个mRNA的5'端，在锥虫中mRNA的生物合成中起关键作用。SL RNA的帽结构在真核生物中是独特的，在帽之后具有4个核苷酸，携带总共7个甲基，并且按照惯例被称为“帽4”。虽然酶机制的帽子添加已被表征在几种生物体，包括布氏锥虫，甲基转移酶的鉴定专用于产生更高的顺序帽结构已经落后，除了在病毒。在这里，我们描述T。布氏杆菌MT57（TbMT 57）是一种主要的核多肽，与牛痘病毒VP 39具有结构和功能相似性，是一种在mRNA 5'端作为帽特异性2'-O-甲基转移酶起作用的双功能蛋白。通过RNAi或TbMT 57的遗传消融的下调导致在位置+3和+4处缺失2 '-O-甲基的SL RNA的积累，因此具有帽2而不是帽4。此外，竞争性结合研究表明，+3和+4位的修饰对于与核帽结合复合物的结合是重要的。MT57的基因切除导致活细胞在SL RNA反式剪接中没有明显缺陷，这表明MT57不是必需的，或者锥虫已经开发了替代机制来抵消这种蛋白质的缺失。有趣的是，MT57同源物仅在具有帽4结构的锥虫原生动物和痘病毒中发现，其中牛痘病毒是原型。
The spliced-leader (SL) RNA plays a key role in the biogenesis of mRNA in trypanosomes by providing the m(7)G-capped SL sequence to the 5' end of every mRNA. The cap structure of the SL RNA is unique in eukaryotes with 4 nucleotides after the cap carrying a total of seven methyl groups and by convention is referred to as "cap 4". Although the enzymatic machinery for cap addition has been characterized in several organisms, including Trypanosoma brucei, the identification of methyltransferases dedicated to the generation of higher order cap structures has lagged behind, except in viruses. Here we describe T. brucei MT57 (TbMT57), a primarily nuclear polypeptide with structural and functional similarities to vaccinia virus VP39, a bifunctional protein acting at the mRNA 5' end as a cap-specific 2'-O-methyltransferase. Down-regulation by RNAi or genetic ablation of TbMT57 resulted in the accumulation of SL RNA missing 2'-O-methyl groups at positions +3 and +4 and thus bearing a cap 2 rather than a cap 4. Furthermore, competitive binding studies indicated that modifications at the +3 and +4 positions are important for binding to the nuclear cap-binding complex. Genetic ablation of MT57 resulted in viable cells with no apparent defect in SL RNA trans-splicing, suggesting that MT57 is not essential or that trypanosomes have developed alternate mechanisms to counteract the absence of this protein. Interestingly, MT57 homologs are only found in trypanosomatid protozoa that have a cap 4 structure and in poxviruses, of which vaccinia virus is a prototype.