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.

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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.