2020 Fiscal Year Research-status Report
Study on molecular mechanism of anti-tumor activity by splicing modulation
| Project/Area Number |
20K15420
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
ChhipiShrestha Jagat 国立研究開発法人理化学研究所, 環境資源科学研究センター, 特別研究員 (40851233)
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| Project Period (FY) |
2020-04-01 – 2022-03-31
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| Keywords | splicing modulation / Next gen sequencing |
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| Outline of Annual Research Achievements |
Splicing modulation by SSA produced the new class of chimeric-intron proteins. A considerable number of intron retained mRNAs were accumulated by SSA treatment, which generate C-terminally truncated exon-intron chimeric proteins through translation until the premature termination codon (PTC) in retained intron as clearly observed in ribosome profiling. Its interesting that those candidates are not targeted by Nonsense mediated decay (NMD) pathway. The most plausible reason for NMD resistance may be premature cleavage and polyadenylation (PCPA) that skips the presence of downstream exon junction complex (EJC) on the splice sites.
Nascent proteome assessment by BONCAT mass spectometry validated the substantial number of exon-intron chimeric proteins. The interesting properties of those chimeric-intron proteins are presence of enriched Intrinsically Disordered Regions (IDRs) and their condensate potential. These aggregation prone intron proteins were observed to be proteotoxic to the tumor cells.
Notably, the global translation was decreased by SSA mediated splicing modulation due to deactivation of mTORC1 which is one of the major axis of tumorigenesis. Along with the global translation downregulation, many tumor specific genes were translationally downregulated. The cumulative results indicated the possibility of neo-proteins from intron source gaining function to selectively inhibiting the growth of tumor cells. Further study in other cancer cells and splicing modulators will clarify the general implication of mechanism discovered upon splicing modulation.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Due to the late availability of other cancer cells and next generation sequencing sending to Berkeley taking longer time, I could not generalize this mechanism showed by splicing modulation yet in highly sensitive cell lines such as leukemia cells.
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| Strategy for Future Research Activity |
Splicing factor mutated cancer cells such as Acute Myeloid leukemia (AML) or Chronic Myeloid Leukemia (CML) were much sensitive to chemical splicing modulation. It is still the subject of the study that how does chemical splicing modulation selectively kill AML or CML cells. Hypothesizing increased intron retention causing increased proteotoxic intron-chimeric proteins and higher mTOR inhibition in AML/CML cells, I plan to carry out system-wide intron retention and intron translation assessment in splicing factor mutated leukemic cancer cells by RNA-seq, ribosome profiling and BONCAT mass spectometry as I have done for HeLa S3 cells. I will further investigate the global translation change and mTOR inhibition upon SSA treatment in range of different cancer cells including these splice factor mutated cancer cells. I hypothesize that the level of intron retention and translation would be correlated to the sensitivity of cancer cells to chemical splicing modulation.
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| Causes of Carryover |
Due to COVID-19, many works were not smooth and as expected.
Purchase of the reagents, cell line and sequencing facility takes the longer time than expected. Hence, next year mainly the remaining budget will be used to buy cell lines, reagents, and sequencing along with publication.
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