| 研究実績の概要 |
We previously identified cytoplasmic capping enzyme (TbCe1) that possess a novel 5′-RNA kinase and guanylyltransferase activities that converts uncapped pRNA into a capped Gppp-terminated RNA. We hypothesize that TbCe1 act on mRNA that has undergone decapping in the cytoplasm to regenerate the functional mRNA. In trypanosome, all decapped mRNA would likely possess a hypermethylation at the 5’-end of the pRNA, which includes dimethyladenosine at position A, uracil at position 4, and 2′-O methylations at position 1, 2, 3 and 4 derived from cap 4 structure.
During the first year of the funding period, we showed that TbCe1 RNA kinase activity can stimulated by the hypermethylation at the 5’-end of the RNA. In particular, dimethyladenosine and 2′-O methylations at position 1 were important for the activity. We also demonstrate that TbCmt1, a second guanine-N7 methyltransferase encoded by trypanosome, is localized in the cytoplasm and its activity can be upregulate by hypermethyation on the RNA, implies that TbCmt1 likely act together with TbCe1 to generate a translatable m7G-capped mRNA. Together these results establish that mRNA recapping enzyme can specifically recognize target mRNA through cap 4 specific methylation. Differential cap methylation may serve as a signal to determine the fate of transcripts to be decapped or recapped.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
As described above, we will continue to make progress on the functional analysis of T.brucei RNA recapping apparatus. In particular, our effort focus to examine the effect of hypermethylated RNA on the m7G RNA methylatransferase activity and to determine the spliced leader RNA sequence specificity on recapping.
We will also continue to explore to identify the uncapped mRNA targets from TbCe1 knockdown strain. We recently optimized the ligation mediated RT-PCR procedure which will be published in Method in Molecular Biology. However, we have encountered technical problems in cloning the target PCR fragments the TA-cloning method, which we aim to resolve this term.
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| 今後の研究の推進方策 |
There will be no major change in the proposed project at this time. We intend to report on our finding on the effect on cap 4 methylation on recapping activities described in aim 2 in form of publication this term. As for the Aim 1 of the project to identify the putatively uncapped transcripts, we are taking an alternative approach to directly sequence the PCR products by next generation sequencing. Once we identify set of mRNA that are potentially regulated by recapping, we intend to examine its mRNA decay properties in the wild-type and the knock-down cells.
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