論文要旨・審査の要旨

学位論文の内容の要旨
論文申請者氏名
安健博
主査：澁谷浩司
論文審査担当者
副査：鍔田武志
副査：上阪等
論文題目
IκBL is a new clue involved in the regulation of alternative splicing in
human and viral genes
（論文内容の要旨）
Abstract
Inhibitor of κB-like protein (IκBL), encoded by HLA-linked NFKBIL1, is a protein of unknown
function, while genetic variations in NFKBIL1 are known to associate with the susceptibility to
inflammatory and/or autoimmune diseases. In this study, it was found that IκBL suppressed exon
exclusion in alternative splicing of human immune-related genes. Yeast-two-hybrid screening and
immunoprecipitation assay revealed molecular association of IκBL with CLK1, a serine/threonine and
tyrosine kinase, which plays a role in the alternative splicing. Unexpectedly, it was found that the
regulation of alternative splicing in CD45 by IκBL was independent from the kinase activity of
CLK1. On the other hand, it was demonstrated that a SR protein, ASF/SF2, bound both IκBL and
CLK1 at the RNA-recognition motifs of ASF/SF2, implying a competition of IκBL and CLK1 on SR
protein. In addition, IκBL was found to regulate the CLK1-dependent synthesis of M2 RNA, a splice
variant of influenza A virus M gene. These observations suggest a functional involvement of IκBL in
the regulation of alternative splicing in both human and viral genes, which is a novel link of HLA
-1-
locus to the regulation of immunity and infection in humans.
Introduction
A number of studies have demonstrated the association between genetic variations in NFKBIL1 and
susceptibility to inflammatory and/or autoimmune diseases. It has been reported that the sequence
variations in the promoter region of NFKBIL1, which showed the lowest and the highest promoter
activity, would confer the susceptibility to rheumatoid arthritis (RA) and Takayasu’s arteritis,
respectively, implying that the altered expression of IκBL might contribute to the pathogenesis of
immune-related diseases.
Pre-mRNAs of human immune-related genes are known to undergo extensive alternative splicing.
Expression of each isoforms generated by the alternative splicing is strictly regulated, whereas the
abnormal alternative splicing events, which lead to altered expression of target mRNA isoforms, have
been reported to associate with autoimmune diseases.
IκBL possesses nuclear localization sequences (NLS) at its N-terminus and localized in nuclear
speckles. In addition, IκBL was reported to associate with RNA. These lines of evidence suggest that
IκBL might play a role in the RNA splicing. I report here that IκBL physically interacts with CLK1
and SR protein, and functions as a novel regulator in the alternative splicing of both human and viral
genes.
-2-
Materials and Methods
Localization of IκBL was examined in cells transfected with EGFP-IκBL. To explore the effects of
IκBL in alternative splicing of human immune-related genes, mini-gene constructs of human CD45,
CD72, or CTLA4, and expression vectors encoding IκBL and related splicing factors were introduced
into cells. Total cellular RNA was harvested and subjected to RT-PCR to analyze the alternative
splicing products derived from the mini-genes. To identify IκBL-interacting proteins,
yeast-two-hybrid screening (Y2H) and immunoprecipitation (IP) assays were applied. The alternative
splicing of endogenous CD45 was examined by RT-PCR and flow cytometry analysis in JSL cells
with or without exogenous IκBL. A plasmid-based rescue system of influenza A virus was employed
to study the impact of IκBL on alternative splicing of influenza M gene.
Results
IκBL-linked EGFP signal was co-localized with SC35, a member of SR protein family, in the
nuclear speckles. In the hnRNPLL-induced CD45 alternative splicing from a mini-gene construct,
IκBL decreased the generation of short isoform and oppositely increased the long isoform. In
addition, IκBL also suppressed the hnRNPLL-induced alternative splicing of CD72, and
FOX1-induced alternative splicing of CTLA4 in the mini-gene assay system. In contrast, knockdown
of NFKBIL1 increased the short isoform and concomitantly decreased the long isoform of CD45,
indicating that IκBL facilitated the exon inclusion in alternative splicing.
A total of 11 different IκBL-interacting proteins, including CLK1, were picked-up by the Y2H
-3-
screening, and the interaction of IκBL with CLK1 was confirmed by IP assay. Knockdown of CLK1
impeded hnRNPLL-induced alternative splicing of both CD45 and CD72 in the mini-gene assay
system, as similar to the inhibition by IκBL, suggesting that IκBL might interfere with CLK1. It was
confirmed that the kinase domain was indispensable for CLK1 to phosphorylate ASF/SF2. However,
IκBL failed to affect the CLK1-induced phosphorylation of ASF/SF2. Both IκBL and CLK1 bound
ASF/SF2 at the RNA-recognition motifs (RRMs).
Overexpression of IκBL in JSL1 cells impaired PMA-induced alternative splicing of endogenous
CD45, and IκBL regulated the splicing of influenza M gene.
Discussion
In this study, IκBL was demonstrated to modulate alternative splicing of various immune-related
genes, suggesting a role of IκBL in a broad context of splicing regulation. CLK1 was identified as an
IκBL-interacting protein. However, IκBL failed to alter the CLK1-induced phosphorylation of
ASF/SF2, indicating a novel mechanism of alternative splicing, where IκBL was involved in, which is
independent from the kinase activity of CLK1.
ASF/SF2 was found to regulate the alternative splicing of CD45, in which the RRMs played an
indispensable role. Because both IκBL and CLK1 associated with the RRMs of ASF/SF2, it was
supposed that IκBL and CLK1 competitively interacted with the RRMs of ASF/SF2 to modulate the
splicing of CD45.
The role of IκBL in alternative splicing of endogenous CD45 implied that PMA-induced splicing of
-4-
CD45 was mediated in part by the reduced expression of IκBL, suggesting that the lower expression
of IκBL could modulate the activation of T cells and hence would be associated with the susceptibility
to RA.
In addition, IκBL was suggested to control influenza viral replication by regulating the level of M2
RNA transcript. This is a so far unraveled mechanism for fighting against invading microorganisms;
by regulating alternative splicing of target viral genes by the HLA-linked gene, NFKBIL1.
In summary, IκBL, which interacts with CLK1 and SR proteins in the nuclear speckles, is one of
the factors playing crucial roles in the alternative splicing in both human and viral genes.
-5-
学位論文の審査の要旨
論文提出者氏名
論文審査担当者
論
文
題目
安健博（甲第4572号）
主査：
澁谷
浩司
副査：
鍔田
武志
副査：
上阪
等
IκBL is a new clue involved in the regulation of alternative splicing in
human and viral genes
（論文審査の要旨）
申請者は自己免疫疾患に関連する NFKBL1 遺伝子である Inhibitor of kB-like protein (IkBL)に着目
し、機能解析を進めた。IkBL が RNA スプライシングに関わる nuclear speckle に局在すること、CD45
遺伝子など免疫に関わるいくつかの遺伝子やインフルエンザウイルスの選択的スプライシングの制御
に関わること、RNA スプライシングに関わる SR タンパクや CLK1 キナーゼに結合し、スプライシング制
御に関与することを初めて明らかにした。
予備審査で付された疑問点、修正点について追加実験を含め、概ね妥当と思われる解答をし、提出された博
士論文において修正が為され、概ね改善されたものとなった。Introduction や discussion に自己免疫疾患
に関連する記述不足が指摘されたものの一連の機能解析は新規性を含め、高いレベルの研究成果と思
われた。したがって、博士学位論文として評価できる。

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