2020 Fiscal Year Research-status Report
Sensitive and highly multiplexed assessment of lncRNA interactions with chromatin
| Project/Area Number |
19K16098
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
柴山 洋太郎 国立研究開発法人理化学研究所, 生命医科学研究センター, 研究員 (60815819)
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| Project Period (FY) |
2019-04-01 – 2023-03-31
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| Keywords | noncoding RNA / long noncoding RNA / chromatin / cell fractionation / RNA sequencing / CAGE sequencing / RNA localisation / RNA displacement |
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| Outline of Annual Research Achievements |
To check the association of noncoding RNA with chromatin, fibroblasts in culture were treated with seven drugs that perturb different pathways in the cell. A comparison of RNA sub-cellular localisation under such stimulated cell state with that under the steady state would allow the functional characterisation of noncoding RNAs by identifying those whose localisations change under different cell states. Following perturbation, cells were fractionated into chromatin, nucleoplasmic and cytoplasmic fractions, and RNA from each fraction was sequenced by CAGE. A computational pipeline for identifying RNA that change compartments upon cell perturbation was created. Surprisingly, drug-specific patterns of RNA displacement was observed, including noncoding RNA shifting towards chromatin.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Both CAGE sequencing and production of a robust computational pipeline to identify RNA that alter sub-cellular localisation and associate with chromatin, have taken more time than initially planned. Low Quantity (LQ) CAGE was undertaken to accommodate large sample size (seven perturbing agents and three control conditions with three sub-cellular fractions in duplicates). This LQ CAGE library preparation was a large undertaking. The computational pipeline is first-of-a-kind and required many trials and careful considerations so that observed results were not analytical artefacts.
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| Strategy for Future Research Activity |
Next step will be validation of sequencing results using RNA smFISH. RNAs which display large displacement will be selected from the sequencing data and will be imaged under the microscope. This will test how good the computational pipeline is. In addition, functional assays for RNAs that shift localisation to chromatin will be done. These assays will show the involvement of those RNAs in the pathways which were perturbed by the drugs.
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| Causes of Carryover |
Validation experiments could not be done in fiscal year 2020 due to sequencing and pipeline production taking more time than initially planned. The amount will be used for the validation experiments in fiscal year 2021.
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