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.

在生长因子/细胞因子、血清或其他因素的刺激下，某些基因被激活，以触发细胞增殖、分化、凋亡和/或细胞运动。这些基因被称为即刻早期基因(IEGs)，是快速和瞬时诱导的。THOC5是转录/输出(TREX)复合体的一个亚复合体，是THO复合体的一员，在RNA的加工、mRNA的输出和基因组的稳定性中起着重要的作用。我们之前已经证明，小鼠体内THOC5的缺失会损害胚胎发育、造血和上皮细胞分化。然而，THOC5的缺失影响了不到1%的基因在稳定状态下的表达。我们最近发现，当THOC5耗尽时，超过90%的血清诱导的IEG mRNAs的3‘加工受到损害。在这种情况下，当THOC5耗尽时，切割和多聚腺苷酸化特异性因子(CPSF)100，一个3‘加工复合体的成员，不能被招募到THOC5靶基因的3’端。此外，我们还发现，在THOC5依赖的IEG mRNAs的多聚腺苷基化后，THOC5与未剪接和剪接的THOC5靶mRNAs结合，然而，THO复合体的分子功能在很大程度上仍不清楚。在这个项目中，我们计划详细研究THO复合体在IEG反应中的作用，并确定参与IEG 3‘加工的蛋白质。我们首先通过芯片测序检测THOC5、CPSF100和其他3’加工复合体的成员，如切割刺激因子kDa(CstF64)和切割因子I(CFIm)在血清诱导的IEG上的分布，并在有或没有THOC5的血清刺激下鉴定CPSF100结合伙伴。CPSF100的血清可诱导结合伙伴的作用将通过与THOC5的免疫共沉淀和使用新的结合伙伴的芯片分析来检验。此外，还将检测潜在结合伙伴基因缺失细胞的IEG反应。此外，我们将研究THOC5和RNA的相互作用结构域，并通过RNA测序识别和表征与THOC5结合的RNA。除了上述在有丝分裂中的作用外，THOC5还可能参与减数分裂过程中程序性DNA双链断裂和基因组稳定性。我们发现THOC5在睾丸中被毛细血管扩张共济失调突变(ATM)激酶高度表达和高度磷酸化。在此背景下，我们将使用条件THOC5基因敲除系统来研究精子发生过程中的这些事件。

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.