The roles of THOC 5/Fms interacting protein, a member of mRNA export complex THOC, in the immediate-early gene expression induced by a cell differentiation signal.

在用生长因子/细胞因子、血清或其他因子刺激时，某些基因被激活以触发细胞增殖、分化、凋亡和/或细胞运动。这些基因被称为立即早期基因（IEGs），它们被快速和短暂地诱导。THOC 5是THO复合物的成员，是转录/输出（TREX）复合物的亚复合物，在RNA的3 ′加工，mRNA输出和基因组稳定性中发挥作用。我们先前已经表明，小鼠中的TH 0 C5的耗尽损害胚胎发育、造血和上皮细胞分化。然而，TH 0 C5的缺失在稳定状态下影响不到1%的基因的表达。我们最近发现，超过90%的血清诱导IEG mRNA的3 ′加工在THOC 5耗尽后受损。在这种情况下，当耗尽THOC 5时，切割和多聚腺苷酸化特异性因子（CPSF）100（3 '加工复合物的成员）不能被募集到THOC 5靶基因的3'末端。此外，我们发现，在THOC 5依赖性IEG mRNA的多聚腺苷酸化后，THOC 5结合到未剪接和剪接的THOC 5靶mRNA，然而，THO复合物的分子功能的细节在很大程度上仍然未知。 在这个项目中，我们计划详细研究THO复合物在IEG反应中的参与，并鉴定参与IEG 3 ′加工的蛋白质。我们首先通过CHIP测序检测THOC 5，CPSF 100和3 ′加工复合物的其他成员，如切割刺激因子64 kDa（CstF 64）和切割因子I（CFIm）在血清诱导的IEG上的分布，以及在存在或不存在THOC 5的情况下，在血清刺激后鉴定CPSF 100结合配偶体。CPSF 100的血清可诱导结合配偶体的作用将通过与THOC 5的免疫共沉淀和使用新结合配偶体的CHIP测定来检查。此外，将检查潜在结合伴侣基因缺失细胞中的IEG应答。此外，我们将检查的相互作用域的THOC 5和RNA，并确定和表征RNA结合的血清模拟后，通过RNA测序。除了在有丝分裂如上所述的作用，THOC 5可能参与程序性DNA双链断裂和减数分裂过程中的基因组稳定性。我们发现，在睾丸中，THOC 5被共济失调毛细血管扩张突变（ATM）激酶高度表达和高度磷酸化。在这种情况下，我们将检查这些事件在精子发生过程中使用条件性THOC 5敲除系统。

Upon stimulation with growth factors/cytokines, serum, or other factors, certain genes are activated to trigger cell proliferation, differentiation, apoptosis and/or cell movement. These genes, called immediate early genes (IEGs), are rapidly and transiently induced. THOC5, a member of the THO complex which is a subcomplex of the transcription/export (TREX) complex plays a role in the 3´processing of RNA, mRNA export, and genome stability. We have previously shown that depletion of THOC5 in mice impaired embryonic development, hematopoiesis, and epithelial cell differentiation. However, the deletion of THOC5 influences expression of less than 1% of genes in the steady state. We recently found that the 3´processing of more than 90% of the serum induced IEG mRNAs is impaired upon depletion of THOC5. In this context, upon depletion of THOC5, a cleavage and polyadenylation specificity factor (CPSF) 100, a member of the 3´processing complex, fails to be recruited to the 3´end of the THOC5 target genes. In addition, we found that after polyadenylation of THOC5 dependent IEG mRNAs, THOC5 binds to unspliced and spliced THOC5 target mRNAs, however, the details of the molecular function of the THO complex are still largely unknown. In this project, we plan to study the involvement of the THO complex in the IEG response in detail and to identify proteins that are involved in 3´processing of IEG.We first examine the distribution of THOC5, CPSF100, and other members of 3´processing complexes, such as Cleavage stimulation factor 64 kDa (CstF64), and cleavage factor I (CFIm) on serum induced IEGs by CHIP-sequencing, and identification of the CPSF100 binding partners upon serum stimulation in the presence or absence of THOC5. The role of the serum inducible binding partner of CPSF100 will be examined by co-immunoprecipitation with THOC5, and by CHIP assay using a new binding partner. Furthermore, IEG response in potential binding partner gene depleted cells will be examined. Furthermore, we will examine the interaction domain of THOC5 and RNA, and identify and characterize RNAs that bind to THOC5 upon serum simulation by RNA-sequencing.In addition to the role in mitosis as described above, THOC5 may be involved in programmed DNA double strand breaks and genome stability during meiosis. We have found that THOC5 is highly expressed and highly phosphorylated by Ataxia telangiectasia mutated (ATM) kinase in testis. In this context, we will examine these events using a conditional THOC5 knockout system during spermatogenesis.