CLOCK interacts with RANBP9 and is involved in alternative splicing in spermatogenesis.
CLOCK interacts with RANBP9 and is involved in alternative splicing in spermatogenesis.
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DOI：
10.1016/j.gene.2017.11.007
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发表时间：
2018-02
期刊：
影响因子：
3.5
通讯作者：
Junbao Yang;Zhi-wei Zhang;Ying-ying Zhang;Xulei Zheng;Yi-lu Lu;D. Tao;Yunqiang Liu;Yongxin Ma
中科院分区：
文献类型：
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作者：
Junbao Yang;Zhi-wei Zhang;Ying-ying Zhang;Xulei Zheng;Yi-lu Lu;D. Tao;Yunqiang Liu;Yongxin Ma
昼夜节律核心基因CLOCK在调节雄性生殖过程中起重要作用。然而，其内在机制仍不清楚。本研究以CLOCK PAS A结构域的cDNA片段为诱饵进行酵母双杂交筛选，发现RANBP 9是一个与CLOCK相互作用的新蛋白。体内和体外实验进一步验证了CLOCK和RANBP 9之间的相互作用。已有研究证实SF 3B 3是一种与RANBP 9相互作用的蛋白。随后，我们的研究还发现CLOCK和SF 3B 3在小鼠睾丸中可以通过免疫共沉淀相互作用。为了研究CLOCK在精子发生中的作用机制，我们还在小鼠睾丸中进行了RNA免疫沉淀和高通量测序（RIP-Seq）。序列分析和Gene Ontology富集分析（生物学过程）结果表明，CLOCK可以直接结合小鼠精子发生中的186个关键mRNA转录本。综上所述，我们的研究结果首次表明，CLOCK可以与RANBP 9相互作用并与mRNA结合，证明CLOCK参与精子发生中的选择性剪接。这些结果揭示了CLOCK在精子发生中的新机制。
The core circadian geneCLOCKplays an important role in regulating male reproduction. However, the underlying mechanism still remains unclear. In the present study, we executed yeast two-hybrid screening using cDNA fragment of CLOCK PAS A domain as bait, and identified RANBP9 as a novel protein interacting with CLOCK. The interaction between CLOCK and RANBP9 was further validated byin vivoandin vitroassays. Previous studies have confirmed that SF3B3 was a RANBP9 interacting protein. Subsequently, our study also found that CLOCK and SF3B3 can interact with each other by co-immunoprecipitation in mouse testis. In order to dissect the underlying mechanism of CLOCK in spermatogenesis, we also performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in mouse testis. The result of sequence analyses and Gene Ontology enrichment analyses (biological processes) demonstrated that CLOCK can directly bind 186 key mRNA transcripts in mouse spermatogenesis. Taken together, our results firstly showed that CLOCK can interact with RANBP9 and bind with mRNAs, demonstrating that CLOCK is involved in alternative splicing in spermatogenesis. These results reveal a novel mechanism for CLOCK in spermatogenesis.