2017 Fiscal Year Annual Research Report
Mechanism of mRNA Recapping pathway in Trypanosome
| Project/Area Number |
16H05180
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| Research Institution | University of Tsukuba |
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Principal Investigator |
Ho Kiong 筑波大学, 医学医療系, 准教授 (20598502)
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| Project Period (FY) |
2016-04-01 – 2020-03-31
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| Keywords | Trypanosome / RNA Capping / RNA processing / RNA modification |
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| Outline of Annual Research Achievements |
Maturation of T.brucei mRNA is achieved by acquiring hypermethylated cap 4 spliced leader RNA by trans-splicing. If recapping enzyme acts on mRNA that has undergone decapping, the physiological substrate would likely preserve all seven methylations at the 5’-end. We showed that conversion of pRNA to GpppRNA can be stimulated by hypermethylation at the 5’-end. During this term, we further characterized the m7G methyltransferase component of recapping enzyme. We found that methyltransferase activity is also stimulated by RNA methylations, but the pattern of methylations differ from the one that activates the RNA kinase activity . These results suggest that cap 4 could serve as a chemical landmark for decapped mRNA to be recapped. Differential methylation patter at the 5’-end could potentially regulate the amount of translatable mRNA.
In addition to RNA recapping, uncapped RNA is a potential substrate for RNA ligase, which can join the 5’-phosphate end with the 3’-hydroxyl end of the RNA. We report that ATP-dependent RNA ligase is capable of removing adenosine from the 3’-end of RNA, generates 2’-3’ cyclic phosphate end at the 3′-terminus. Such a modification could prevent the ligation and could allow RNA to be recapped in the cells. We published these findings in Scientific Reports.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The aim 1 of this project is to explore the mRNA targets for recapping in T.brucei. While we have modified the protocol for identifying the physiological RNA substrate for recapping through Ligation Medicated RT-PCR, we encountered difficulties identifying uncapped transcripts that are specifically accumulated in recapping enzyme depleted cells due to a high background level. We have since altered the approach and examining a change to uncapped transcripts population by Next Generation Sequences using Nanopore technology.
We have essentially completed the aim 2 of the project as proposed and are readying for publication.
We have initiated aim 3 of the project to examine whether hypermethylation could affect the mRNA turnover as described below.
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| Strategy for Future Research Activity |
As proposed in Aim 3, we hypothesize that methyl moieties that are retained at the 5′-end of the decapped transcript could possibly regulate RNA degradation. Preliminary result using the commercial 5’-exonuclease suggests that methylations could inhibit the 5’-3’ exonuclease activity and therefore allows uncapped transcripts to accumulate in the cell for recapping. During this term, we will focus to evaluate what type of methylation could protect pRNA from exonuclease digestion. We will attempt to express and purify the four trypanosome 5’-exonuclease and examine whether hypermethylation could affect the nuclease activities.
We will also continue to explore to identify mRNA targets for recapping in T.brucei.
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