「様式３－B」

課題番号（Project number）25

Mouse model for nucleolus instability leading to early aging:
RACK1-APNX-PARP1 complex［１］組織（Research group）
代表者（Principal Investigator (PI)）：
Gijbertus T.J. van der Horst（Erasmus University Medical Center）
対応者（Host researcher at IDAC）：
Akira Yasui（IDAC, Tohoku University）
分担者（Co-investigator）：
Shin-ichiro Kanno（IDAC, TohokuUniversity）
研究費 (Expenditure report of research funds)：
物件費 200,000YEN，旅費 0 YEN［２］研究経過（Research setup）
Human APNX gene has been isolated, but
unpublished yet, by Shinichiro Kanno using
bioinformatics of PARP1-binding DNA repair
protein PALF (Kanno et al. EMBO J. 26,2094-2103, 2007). By immune-precipitation
experiments it turned out that APNX builds aheterodimer with PARP1 and knock down of
APNX expression provided cells with sensitivity
to MMS, suggesting its role in the response tooxidative DNA damage. PARP1 is activated byDNA single-strand break (SSB) or DNA
double-strand
break(DSB)
andpoly(ADP-ribosyl)ated itself and its surrounding
proteinsneartheDNAdamage.
Poly(ADP-ribosyl)ation acts as a signal for DNA
damage and many DNA repair proteins as well as
RNA-relared proteins accumulate at thepoly(ADP-ribose). There are many proteins withRNA interacting domain designated as
intrinsically disorder proteins (IDPs) with low
complexity domains like Fus, EWS, TARF15 andRBM14. These proteins form foci, called as stressgranule in response to oxidative stress and arebelieved to function as RNA maintenance andpossibly in DNA repair. We have previously
identified RACK1 protein as an interacting

protein of RECQL4, mutations in which areassociated with the autosomal recessive disease
Rothmund-Thomson Syndrome, a disorder thathas features of premature aging. RACK1
interacts with IDPs and forms stress granule inresponse to H2O2 treatment of cell. We here
analyzed interaction between RACK1 and APNX,
both of which respond to DNA damage andPARP1 dependent poly(ADP-ribosyl)ation.
Akira Yasui visited Erasmus University
Medical Center Rotterdam twice in 2016,
February and September, and we discussed thissubject thoroughly.
Because of the preparation for AAALAC
application, mouse model has not yet beencreated. Instead we have analyzed and identified
the interaction between APNX and RACK1
shown below.
［３］成果（Research outcomes）（３－１）研究成果（Results）
We have first characterized GFP-tagged RACK1
expressedinU2OS
cellwithlaser
micro-irradiation (Fig. 1). GFP-tagged RACK1
accumulated to laser-irradiated site and indicatedits response to DNA damage. We then establisheda HEK293 cell line having integrated andconstitutively expressing FLAG-tagged RACK1
gene. By immune-precipitation using FLAG
antibody we pulled down TRACK1 interacting
proteins (Fig. 2). We identified APNX interactingwith RACK1 in oxidative stress-dependent
manner. Thus, we identified RACK1, which
contributes to DNA repair by interacting withDNA repair protein and endonuclease APNX inresponse to oxidative DNA damage.

Figure 1. Accumulation of GFP-Tagged RACK1
at laser-irradiated site.

Figure 2. RACK1 interacting proteins i（３－２）波及効果と発展性など（Futureperspectives）
APNX and PARP1 are found in the nucleolus andinteract to each other. RACK1, however, ispresent in the nucleus but not in the nucleolus.
Therefore, in response to oxidative stress, RACK1
may build a complex with APNX and PARP1
enabling DNA damage repair. Since oxidative
DNA damage plays the most important role ingenome integrity, APNX-PARP1-RACK1 complex
may be a core complex for anti-cancer andanti-aging.

［４］成果資料（List of Papers）
Manuscript is in preparation.