研究題目 Proteomics analysis of recombination complex that
promotes site specific genomic instability in B cell
研究組織
研究代表者：本庶 佑（京都大学大学院医学研究科）
共同研究者：小迫 英尊（徳島大学先端酵素学研究所）
研究分担者：Nasim Begum（京都大学大学院医学研究科）

小林牧（京都大学大学院医学研究科）【1】研究の概要[1-1] 本研究の目的・概要・ Purpose and outline
Antigen activated mature B cells undergo Ig gene diversity through a complex DNA break
and recombination mechanism induced by Activation-Induced Cytidine Deaminase (AID).
In order to understand the AID induced this recombination mechanism, we need tocharacterize (1) the RNA/DNA editing protein complex that associates with AID and its cofactor, and (2) the chromatin complex that accumulates at the Ig locus during antibody
class switch recombination (CSR).
One of the AID’s co-factor for CSR is hnRNPL, which interacts with AID and otherunknown proteins during CSR. Recently, mutagenesis study identified critical hnRNPL
mutants that are unable to support CSR. Therefore, the specific purpose is to know theproteins interacting with wild type-hnRPL but not with the CSR loss of function mutanthnRNPL.

[1-2] 研究の方法・経過・Method
Emploing standard mutagenesis procedure, deletion and point mutants of hnRNPL weregenerated. CSR efficiency of various mutants was examined by monitoring IgM to IgA
switching efficiency in a mouse B cell line. WT and mutant proteins were also examined
for their interaction with AID in 293T cells. Since hnRNPL was tagged with FLAG epitope,
we performed anti-FLAG immunprecipitation(IP) of WT and the mutant hnRNPL. IPed

proteins were separated on TGX gradient gel and silver stained. The gels with promisingdifference (WT versus Mutant) were sent to Prof. H. Kosako’s laboratory at Tokushima
University for MS analysis.

【2】研究成果[2-1] 本共同研究で明らかになった研究成果・Result

The

schematic view shows the domain structure of wild type and two critical hnRNPLmutants. IgM to IgA switching efficiency was examined, which indicates that both themutants are defective in CSR.

Comparison of hnRNPL immunnoprecipitation on silver stained gel. Red rectangles
indicate the area of interest, which have been subjected to MS analysis.

Many proteins including known AID interacting proteins were identified from thisinitial trial. The Table below (comparison of WT vs. Mut-1) is an example, whichshows that some proteins can be found only with WT but not Mutant-1.

[2-2] 本共同研究による波及効果及び今後の発展性・Discussion
It is surprising that some known AID interacting protein like DHX or THO was detected in
the hnRNPL IP. Moreover, many interacting proteins appear to be RNA binding proteins.
If this is the case, AID-hnRNPL could be a much bigger RNP (ribonucleoprotein) complex
than we expected. Currently, little is known about the function of AID’s RNP complex withhnRNPL. MS results showed some promising interacting molecule like RBM15B, because
another RBM protein (RBM47) has been found as a RNA editing co-factor for Apobec1.

【3】主な発表論文等[3-1] 論文発表
1）[雑誌]著者名.題名.雑誌名

巻：頁‐頁，発行年. (publication in journals)2）[書籍]著者名.題名.Ｉｎ：編集者名・編 書籍名.出版社,発行地，頁-頁,発行
年 (publication in books)[3-2] 学会発表
1）発表者名.題名.学会名，発表地，発表月日，発表年(presentation in meetings)[3-3] 成果資料等【4】今後の課題等 (Future directions)
We need reproducible IP/MS result for hnRNPL from 293T and B cell system. As per Prof.
Kosako’s suggestion gel-free MS approach will be adopted for the faster progress, andalso to obtain a comprehensive overview of the interacting proteins. Under identicalcondition, AID IP will be also necessary in the near future.