| 研究課題/領域番号 |
19K16691
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| 研究機関 | 大阪大学 |
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研究代表者 |
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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| 研究期間 (年度) |
2019-04-01 – 2021-03-31
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| キーワード | immune regulation |
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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
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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| 今後の研究の推進方策 |
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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| 次年度使用額が生じた理由 |
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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