2019 Fiscal Year Research-status Report
GANP may regulate the c-Myc expression in germinal centers (GCs), that is essential for the affinity maturation of B cells
| Project/Area Number |
19K16691
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| Research Institution | Osaka University |
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Principal Investigator |
シン シャイレンドラ・クマール 大阪大学, 免疫学フロンティア研究センター, 特任研究員(常勤) (10792187)
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| Project Period (FY) |
2019-04-01 – 2021-03-31
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| Keywords | immune regulation |
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| Outline of Annual Research Achievements |
We carried out mass spectrometry (MS) analysis of GANP in B cells. I found that GANP directly interacts with the translation initiation components. GANP-RNA binding data suggest that GANP interacts with both the 3’UTRs and 5’UTRs of RNAs. Gene ontology term analysis revealed enrichment of genes involved in cell cycle regulation, DNA repair, helicase activity, translation initiation, and immune regulation.GANP markedly interacts with c-Myc mRNA both in human and mouse B cells. GANP significantly augments the Luciferase activity through interacting with the c-Myc mRNA via 5’UTR. GANP-knockdown in B cells significantly decreased the protein level of c-Myc and Bcl2.
The results suggest that GANP significantly targets the restricted and specified mRNAs such as c-Myc and Bcl-2 in B cells.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
GANP is up-regulated molecule in GC B cells upon T cell dependent immune responses, contains multiple domains having different functions.
In order to investigate the detail molecular function of GANP, I am making several deletion mutants and point mutation at specific functional domain of GANP.
c-Myc 5’UTR having a very complex RNA structure, I am making several mutants at this region and trying to do functional Luciferase assays.
In order to find out the function of GANP in GC b cells, we are making mice to delete the GC specific GANP molecule and analise the detail molecular functions during immune responses.
I am also collecting the immune organ and making the tissue sections slide for immunohistochemistry and Immunofluorescence.
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| Strategy for Future Research Activity |
GC is organized into two major compartment dark zone (DZ) and light zones (LZ), c-Myc expression mainly in the LZ is required for B cell survival. GANP may control the c-Myc mRNA and protein expression together with some binding co factors in GC B cells for affinity maturation and B cell selection.
I would like to demonstrate that, how GANP regulates the c-Myc expression and GC B cells kinetics during immunization (WT/KO/Tg mice), using chromium single cell immune profiling (10X genomics).
Single cell RNA sequence will distinguish the GANP and c-Myc positive population in GC B, DZ and LZ cells among the WT/KO/Tg mice.
I would like to investigate the localization of GANP and its associated co factors at post transcription level using Immunofluorescence (IF)
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| Causes of Carryover |
I would like to demonstrate that, how GANP regulates the c-Myc expression and GC B cells kinetics during immunization (WT/KO/Tg mice), using chromium single cell immune profiling (10X genomics). I would like to prepare the samples according to the mice colony availability and Osaka University RNA sequencing facility. Sample preparation will take few more months that's why I planned to perform the experiments in FY2020.
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Research Products
(2 results)
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[Journal Article] Feedback regulation of Arid5a and Ppar-γ2 maintains adipose tissue homeostasis2019
Author(s)
Chalise Jaya Prakash、Hashimoto Shigeru、Parajuli Gyanu、Kang Sujin、Singh Shailendra Kumar、Gemechu Yohannes、Metwally Hozaifa、Nyati Kishan Kumar、Dubey Praveen Kumar、Zaman Mohammad Mahabub-Uz、Nagahama Yasuharu、Hamza Hanieh、Masuda Kazuya、Kishimoto Tadamitsu
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Journal Title
Proceedings of the National Academy of Sciences
Volume: 116
Pages: 15128～15133
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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