Sensitive and highly multiplexed assessment of lncRNA interactions with chromatin

2021 Fiscal Year Research-status Report

• Project/Area Number: 19K16098
• Research Institution: Institute of Physical and Chemical Research
• 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)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Keywords: lncRNA / subcellular localization / CAGE sequencing / smFISH

Outline of Annual Research Achievements

In the 2020 fiscal year I generated data showing lncRNA subcellularl localization is dynamic upon treatment of the cell with a DNA damage inducing agent. In the 2021 fiscal year, to get a broad overview of how dynamic lncRNA subcellular localization can be, I investigated it at the genome-wide level upon treatment of the cell with seven pathway-defined stress inducers targeting essential cellular processes including transcription, translation and epigenetic modifications. This was done by CAGE sequencing of RNA purified from the chromatin, nucleoplasmic and cytoplasmic fractions of the cell. To analyze the CAGE data I developed a pipeline to effectively identify RNA which re-localizes across three fractions. Surprisingly, lncRNA subcellular localization turned out to be highly dynamic, whereby each stress inducer produced a distinct pattern of re-localization across three fractions. Moreover, different stimuli could cause the same lncRNA to re-localize to different locations. Re-localization of fifteen lncRNAs were verified by single-molecule FISH.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

In the 2021 fiscal year I analyzed a large dataset that resulted from CAGE sequencing, which led to a surprising finding that lncRNA subcellular localization is highly dynamic across various stimulations. I also verified this by smFISH.

Strategy for Future Research Activity

The next and final step of the project is to investigate whether lncRNA re-localization has functional consequences for the cell. To assess this I have chosen two target lncRNAs, Chaserr and MIAT. My data has shown that Chaserr re-localizes from the chromatin to cytoplasm upon stimulation with cycloheximide, a translation elongation inhibitor, whereas MIAT is enriched in the chromatin upon stimulation with valproic acid, an HDAC inhibitor. There is literature suggesting Chaserr can interact with mRNA, so my aim is to identify those interaction partners of Chaserr in the cytoplasm. For MIAT, DNA loci of its activity will be identified. These will be done by ChIRP-seq of Chaserr-interacting RNA and MIAT-interacting DNA.

Causes of Carryover

Due to bioinformatic analyses which took longer than initially anticipated, experimental costs were lower than originally planned. The remaining amount will be used in the next fiscal year, which will involve ChIRP-seq for two lncRNAs. This will show functionality of lncRNA re-localization.