THOC5, a member of the mRNA export complex: a novel link between mRNA export machinery and signal transduction pathways in cell proliferation and differentiation

Abstract

Cell growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of growth factor/cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation have been mainly studied by focusing on the cis- and trans-elements in promoters however, the role of mRNA export machinery during differentiation has not been adequately examined. THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5) is a member of THO complex which is a subcomplex of the transcription/export complex (TREX). THOC5 is evolutionarily conserved in higher eukaryotes, however the exact roles of THOC5 in transcription and mRNA export are still unclear. In this review, we focus on recently uncovered aspects of the role of THOC5 in signal transduction induced by extracellular stimuli. THOC5 is phosphorylated by several protein kinases at multiple residues upon extracellular stimuli. These include stimulation with growth factors/cytokines/chemokines, or DNA damage reagents. Furthermore, THOC5 is a substrate for several oncogenic tyrosine kinases, suggesting that THOC5 may be involved in cancer development. Recent THOC5 knockout mouse data reveal that THOC5 is an essential element in the maintenance of stem cells and growth factor/cytokine-mediated differentiation/proliferation. Furthermore, depletion of THOC5 influences less than 1% of total mRNA export in the steady state, however it influences more than 90% of growth factor/cytokine induced genes. THOC5, thereby contributes to the 3' processing and/or export of immediate-early genes induced by extracellular stimuli. These studies bring new insight into the link between the mRNA export complex and immediate-early gene response. The data from these studies also suggest that THOC5 may be a useful tool for studying stem cell biology, for modifying the differentiation processes and for cancer therapy.

Figure 1

Illustration of domain structure and phosphorylation sites of THOC5. A: Arrangement of potential functional domains in THOC5. NLS: nuclear localization signal; WWB: potential binding site for proteins with WW-domains; PEST: potential PEST domain; LZ: potential leucine zipper; p: phosphorylation sites. Conserved domain (more than 50% amino acid identity in a 10 amino acids or longer stretch) between mouse THOC5 (683 amino acids long: 683AA) and Drosophila melanogaster (Drosophila) (616AA), C. elegans (599AA) or S. pombe (200 AA) THOC5 is shown as grey boxes. Numbers represent amino acid numbers from mouse THOC5. B: An alignment of the key regions among mouse, human, Xenopus tropicalis (Xenopus), Danio rerio, Drosophila, C.elegans and S. pombe.C: Summary table of phosphorylation sites of THOC5.

Figure 2

Subcellular localization of THOC5. A: Schematic representation of subcellular localization and immunostaining of endogenous THOC5. In immature macrophages, THOC5 is located mainly in the nucleus (nuclear speckle-like domains), while in mature macrophages THOC5 is also found in the cytoplasm [20]. Immunostaining: Human mesenchymal stem cells were fixed by methanol and then used for THOC5 specific staining using THOC5 monoclonal antibody [22]. THOC5 is located mainly in the nuclear speckles. B: Nuclear/cytoplasmic shuttling of THOC5 in Hela/mouse 3 T3 cells. Heterokaryon assay: myc-tagged THOC5 was expressed transiently in Hela cells which were then fused with mouse NIH/3 T3 cells in the presence of cycloheximide. Cells were stained by anti Myc-antibody followed by tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti mouse IgG and with 4′,6-diamidino-2-phenylindole (DAPI). *: fused Hela cells; M: mouse NIH /3 T3 cells [23].

Figure 3

Identification of THOC5 dependent genes in steady state and under serum stimulation conditions. A: Summary table of transcriptome analyses in THOC5 depleted Hela cells [32], mouse embryonic fibroblasts [25], and bone marrow derived macrophages [20]. B: Transcriptome analysis of serum inducible genes in mouse embryonic fibroblasts from tamoxifen inducible THOC5 knockout mice [21]. Mouse embryonic fibroblasts with or without THOC5 were serum starved for 24 h and then stimulated with serum for 1 h. Total RNA was isolated for transcriptome analysis as described previously [20]. Ninety-four genes were upregulated more than 4-fold in the presence of THOC5. In absence of THOC5, only 9 genes were induced to a similar extent. * including direct and indirect THOC5 dependent genes.

Figure 4

Potential function of THOC5 in immediate-early gene response. Upon stimulation with growth factors/cytokines THOC5 is phosphorylated at multiple positions. Signals reach the nucleus and the promoters of immediate-early response genes are activated. Simultaneously, the 3′ processing complex is recruited to the promoter region [32]. During gene transcription, THOC5 is recruited to its target immediate-early gene [20] and then participates in 3′ processing of these genes. Unspliced and spliced THOC5 target mRNAs form a complex with THOC5. Post transcriptional splicing occurs in nuclear speckles where THOC5 has accumulated [20]. For the export of spliced mRNAs from nuclear speckles other members of TREX, Aly, UAP56 and THOC2 are required [30,44]. It has been suggested that THOC5 and Aly form a complex with mRNA receptor TAP-p15 transiently and transfer THOC5 target mRNA to the TAP-p15 complex [29]. PM: plasma membrane; NP: nuclear pore.