| 研究実績の概要 |
We showed that: (i) Hypermethylation at the 5’-end of the RNA could stimulate the TbCe1 RNA kinase activity; (ii) Methyltransferase activity of TbCmt1 is also stimulated by RNA methylations, but the pattern of methylations differ from the one that activates TbCe1 RNA kinase activity; (iii) The 2’-O methylation at the 5’-end could inhibit the 5’-3’ degradation to protect the decapped RNA. These findings suggest that RNA methylations derived from cap 4 structure, serves as an imprint for the specific mRNA to be preferentially recapped to regulate the gene expression.
We extended our project to characterize a novel 3’-deadenylation activity by RNA ligase. In this term, we also initiated a project to examine the structure of RNA triphosphatase that catalyzes the 1st step of mRNA capping.
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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
While we have modified the protocol for identifying the physiological RNA substrate for recapping through Ligation Medicated RT-PCR, we still have difficulties identifying uncapped transcripts that are accumulated by depletion of recapping enzyme. In this term, we will focus on evaluating the mRNA expression from recapping enzyme depleted cells to gain insight on what kind of biological pathways.
The RNA triphosphatase is a promising anti-infective drug target against protozoan parasites. We crystallized the trypanosome RNA triphosphatase solve the structure of the enzyme in complex with tripolyphosphate in the active site and proposed a mechanism on how RNA is recognized by the enzyme.
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| 今後の研究の推進方策 |
In this them, we will continue to investigate in identifying the in vivo targets for mRNA recapping, and examining the methylation effect on mRNA turnover. In light of our finding that methylations adjacent to the cap structure is critical for mRNA recapping, we initiated a project to identify the RNA methyltransferase enzyme responsible for cap 4 biosynthesis. Specifically, we are interested in cap-dependent m6A RNA methyltransferase, which was recently in human. We intend to generate conditional knock-outs and knock-downs cap-dependent RNA methyltransferases by an inducible-CRISPR-Cas9 system (T. cruzi) and RNA interference (T brucei), respectively, to evaluate the effects on parasite gene expression.
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