RNA-binding motifs of hnRNP K are critical for induction of antibody diversification by activation-induced cytidine deaminase

全文

(1)

Title

RNA-binding motifs of hnRNP K are critical for induction of

antibody diversification by activation-induced cytidine

deaminase( Abstract_要旨 )

Author(s)

Yin, Ziwei

Citation

京都大学

Issue Date

2020-07-27

URL

https://doi.org/10.14989/doctor.k22698

Right

Type

Thesis or Dissertation

Textversion

ETD

(2)

京都大学

博士( 医科学 )

氏 名

尹 紫薇

論文題目

RNA-binding motifs of hnRNP K are critical for induction of antibody diversification

by activation-induced cytidine deaminase

(論文内容の要旨)

Activation-induced cytidine deaminase (AID) is specifically expressed in activated B lymphocytes and is responsible for class switch recombination (CSR) and somatic hypermutation (SHM) in the adaptive immune system. After activation of B lymphocytes by antigen, AID is expressed and then initiates DNA breaks in both switch (S) and variable (V) regions of immunoglobulin heavy chain (IgH) genes followed by the different repair steps for SHM and CSR, resulting in antibody diversification. However, there has been a long-standing debate regarding the molecular mechanism of AID in DNA breaks in the V and S regions and repair in the S regions. Because AID is the cytidine (C)-to-uracil (U) converting enzyme, the question of which is the target of AID—C in RNA or C in DNA, has not been resolved yet. “DNA deamination by AID” hypothesis proposes that base excision repair or mismatch repair mechanism produces DNA breaks. However, various mutants of AID showed that level of in vitro DNA deamination does not always correlate with the frequencies of SHM and CSR in vivo, questioning the plausibility of DNA deamination by AID. This study was based on “RNA editing” hypothesis, which proposes that AID edits some putative RNAs for DNA breaks and the other RNAs for DNA repair with the help of the several cofactors. Among the AID-cofactors, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was previously identified to function for inducing DNA breaks.

In this study, to answer the question of which domain of hnRNP K is responsible for the association with AID and DNA breaks, the molecular dissection of hnRNP K was investigated by using murine lymphoma-derived CH12F3-2A cells, which enable monitoring of CSR from IgM to IgA by antigen stimulation, as well as AID-dependent DNA breaks and other IgH gene recombination events. Two possible types of RNA-binding domains are found in hnRNP K. The first type is the three K homology (KH) domains, and the other is the K-protein-interaction (KI) domain. Every KH domain contains a GXXG motif, which favorably binds to poly(C) sequences in both RNA and ssDNA. The KI domain encodes multiple RGG motifs, which has the potential to bind both proteins and RNAs. This study showed that both the GXXG and RGG motifs played an important role in CSR and SHM, because they were necessary for AID-dependent DNA breaks. Moreover, CSR- and SHM-deficient hnRNP K mutants almost lost the RNA-dependent interaction with AID, as well as the ability to bind with the typical hnRNP K-binding RNAs. It suggested that specific RNA(s), binding of which was abolished by the mutation of GXXG or RGG motifs, might be responsible for AID-dependent DNA breaks. Additionally, both GXXG and RGG motifs were required for nuclear localization of hnRNP K. Because it was previously shown that nuclear localization signal (NLS) mutants of AID are defective in CSR and SHM, lower nuclear localization in these RNA-binding motif mutants could partially contribute to their malfunction in AID-dependent DNA breaks. These results suggested that hnRNP K presents some RNAs to AID for editing through GXXG and RGG motifs, and the edited RNAs provoke DNA breaks in IgH locus.

Still, the molecular mechanism of AID-dependent DNA breaks is highly enigmatic—which RNA is edited by AID, what is the function of the edited RNA, and how the DNA is cut. However, the remaining questions will be answered when RNAs associated with hnRNP K are analyzed. In the future, comparing the trapped RNAs between the motif mutants and the wild type hnRNP K will give an important clue to the molecular mechanism of AID-induced DNA breaks.

(論文審査の結果の要旨)

Activation-induced cytidine deaminase(AID)は B 細胞に特異的に発現し、抗体遺伝子

における Class Switch Recombination(CSR)と Somatic Hypermutation(SHM)を制御する

ことで抗原排除に有利な抗体を産生する酵素である。CSR と SHM 両過程は AID 依存性の

抗体遺伝子組換え部位の DNA 切断により始まる。先行研究では、RNA 結合タンパク質で

ある heterogeneous nuclear ribonucleoprotein K(hnRNP K)がその DNA 切断に必要であるこ

とを発見した。本研究では、hnRNP K の RNA 結合モチーフである GXXG と RGG に点変

異を導入することで、GXXG と RGG が AID 依存的な抗体遺伝子 DNA 切断と続いて起き

る CSR と SHM、さらに cMyc 遺伝子との転座に必要であることを明らかにした。GXXG

と RGG は hnRNP K の RNA 結合能力に必須であり、AID と hnRNP K との結合にも重要で

あった。また、これらのモチーフは hnRNP K の核内局在も制御した。これらより、hnRNP

K は GXXG と RGG を通して AID に編集 RNA を提示し、編集された RNA が抗体遺伝子

の切断に関わることが予想された。

以上の研究は hnRNP K が抗体多様性に果たす分子メカニズムの解明に貢献し、生体

防御機構の理解に寄与するところが多い。

したがって、本論文は博士( 医科学 )の学位論文として価値あるものと認める。

なお、本学位授与申請者は、令和2年6月1日実施の論文内容とそれに関連した試

問を受け、合格と認められたものである。

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