InteractiveFly: GeneBrief

thoc5: Biological Overview | References

The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. This study shows that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, it was shown that TREX is required for accumulation of nascent piRNA precursors. These data reveal a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis (Hur, 2016).

Thoc5 mRNA loading piRNA biogenesis transcriptional regulation THO is a multiprotein complex, which itself is part of the TREX (transcription/export) complex. The THO complex was first identified in Saccharomyces cerevisiae and is conserved throughout metazoan evolution (Zhou, 2000; Katahira, 2012). In metazoans, THO contains six proteins; three proteins are homologous to yeast proteins (Hpr1/Thoc1, Thoc2, and Thoc3/Tex1), and three are unique (Thoc5/FMIP, Thoc6, and Thoc7) (Aguilera 2005). In addition to the THO subunits, the complete TREX contains two proteins conserved between yeasts and mammals: Yra1, also known as REF/Aly, and Sub2, also known as UAP56. In yeast, the THO/TREX complex participates in transcription, pre-mRNA processing, and nuclear mRNA export of the majority of genes. Yeast TREX associates with transcribing RNA polymerase II (Pol II) and is necessary for transcription elongation. TREX is loaded onto nascent transcripts during transcription and is essential for mRNA export from the nucleus, as the Yra1 subunit of TREX recruits the Mex67p protein, which interacts with nucleoporins and mediates passage of complexes of mRNAs and proteins (mRNPs) through the nuclear pore (Hur, 2016).

Despite the conservation of the THO/TREX complex in yeast and metazoa, there are important differences in its function in different organisms. As it is in yeast, metazoan TREX is loaded onto nascent transcripts; however, it does not associate with RNA polymerase. Instead, association of TREX with pre-mRNA depends on splicing of pre-mRNA (Masuda 2005) and requires the interaction of several TREX subunits with the nuclear cap-binding proteins CBC20 and CBC80 (Cheng, 2006). Most metazoan genes contain introns, leading to effective splicing-dependent TREX loading onto mRNAs. Furthermore, TREX is recruited to several intronless genes in Drosophila and mammals (Nojima, 2007; Kopytova, 2010). For some of these genes, TREX recruitment is mediated by sequence-specific RNA-binding proteins that recognize distinct motifs on the mRNAs (Chi, 2014); in other cases, the mechanism of splicing-independent loading is not understood (Hur, 2016).

The conserved composition of TREX suggests that it plays the same function in the nuclear export of mRNPs in metazoa as it does in yeast. Indeed, in Drosophila, depletion of Sub2/UAP56 causes accumulation of polyadenylated RNAs within the nucleus (Gatfield, 2001). In contrast, however, depletion of other TREX components has a surprisingly mild effect on mRNA export and gene expression in Drosophila and mammals (Gatfield, 2002; Rehwinkel, 2004; Chi, 2013). Depletion of the THO complex in Drosophila S2 cells results in changes in expression of only a small fraction of genes (Rehwinkel, 2004). Similarly, in HeLa cells deficient in Thoc5, no significant accumulation of poly(A) RNAs is observed in the nucleus (Chi, 2013). Transcriptome analyses of mouse embryonic fibroblasts in which thoc5 expression was inhibited showed down-regulation of only 143 genes, and these were efficiently spliced but retained in the nucleus (Guria, 2011). In Drosophila, thoc5 mutants are viable but have spermatogenesis defects (Moon; 2011; Hur, 2016 and references therein).

This study shows that Thoc5 and other THO subunits are required for female fertility, oocyte patterning, and transposable element (TE) repression in the Drosophila germline. Thoc5 is required for biogenesis of piRNAs, a distinct class of small noncoding RNAs (ncRNAs) that are expressed in germ cells and guide transposon silencing. Mature 23- to 28-nucleotide (nt) piRNAs are processed from long, noncoding transcripts (piRNA precursors) generated from distinct genomic regions dubbed piRNA clusters. Most piRNA clusters in the Drosophila genome are transcribed from both genomic strands and are therefore called dual-strand clusters; the rarer unistrand clusters are transcribed from one strand. A group of proteins composed of the HP1 homolog Rhino (Rhi), the RNA helicase UAP56, and two proteins of unknown function, Cutoff (Cuff) and Deadlock (Del), were shown to be essential for piRNA biogenesis from dual-strand but not unistrand clusters, indicating that piRNA biogenesis from these two types of clusters is quite different. Rhi, Del, and Cuff form the RDC complex that associates with chromatin of dual-strand but not unistrand clusters (Le Thomas, 2014; Mohn, 2014; Zhang, 2014). The chromodomain of Rhi directly binds the H3K9me3 mark, which is enriched in the chromatin of dual-strand clusters (Hur, 2016).

Thoc5 localizes in distinct nuclear foci that overlap with sites of piRNA transcription. In addition, it was observed that Thoc5 is highly enriched on noncoding piRNA transcripts compared with protein-coding pre-mRNAs. Binding of Thoc5 to unspliced piRNA precursors and localization to nuclear foci require Cuff protein that is present on the chromatin of piRNA clusters. The data reveal a novel, splicing-independent mechanism of TREX loading on nascent RNA. Thoc5 was also found to be necessary for accumulation of nascent piRNA precursors, indicating that TREX has a role in transcriptional control (Hur, 2016).

This study found that Thoc5 and other components of the THO complex are required for Drosophila ovarian germ cell development and fertility. Further experiments revealed that THO is necessary for repression of multiple TE families. In agreement with othese results, several components of the THO complex were identified in a genome-wide RNAi screen for factors involved in TE repression in germ cells (Czech, 2013; Hur, 2016 and references therein).

TE derepression in Thoc5-deficient ovaries is caused by a decrease in the abundance of piRNAs, ncRNAs that guide transcriptional and post-transcriptional repression of TEs in germ cells. Multiple lines of evidence suggest that THO is directly involved in piRNA biogenesis. First, several THO subunits (Thoc5, Thoc2, and Thoc7) localize at distinct nuclear foci that coincide with Rhi, a factor that is enriched on piRNA clusters and required for piRNA biogenesis (Mohn, 2014). Second, two THO components (Thoc5 and Thoc7) were found to be enriched on long piRNA precursor transcripts and are necessary for their expression (Hur, 2016).

In yeast and mammals, THO, the RNA helicase UAP56 (called Sub2 in yeast), and Aly/Ref together form the TREX complex, which is loaded on nascent RNA. UAP56 is required for piRNA biogenesis and binds to piRNA precursors (Zhang, 2012). This study found that Thoc5 forms a complex with UAP56 in an RNA-independent fashion. Furthermore, Thoc5 and UAP56 colocalize in nuclear foci, and their localization, but not protein stability, is interdependent. These data suggest that a preformed TREX complex is loaded on piRNA precursors. Although TREX is considered to be a general factor, which is loaded on nascent RNA transcribed by RNA Pol II, the current RIP-seq data showed that association of TREX with different nascent transcripts is quantitatively different. Indeed, while <5% of mRNAs display greater than twofold enrichment in Thoc5 RIP, transcripts from all dual-strand piRNA clusters are enriched. Enrichment of TREX on piRNA precursors raises the question about the mechanism for its specific loading to these transcripts. This question is particularly intriguing, since association of TREX with nascent RNAs in metazoa was previously shown to be dependent on splicing (Masuda, 2005), but piRNA precursors are not spliced (Zhang, 2014). Furthermore, splicing of piRNA precursors induced by depletion of Rhi was shown to have a negative effect on piRNA biogenesis (Zhang, 2014). The clue to this puzzle was provided by the fact that both Thoc5 and UAP56 are required for piRNA biogenesis from only a subset of all genomic regions that generate piRNA; namely, dual-strand piRNA clusters. The same exclusive effect on dual-strand clusters was previously shown for Rhi and Cuff, two proteins that form a complex on chromatin of dual-strand clusters due to direct binding of the H3K9me3 histone mark by the chromodomain of Rhi. This study found that TREX and Cuff physically and genetically interact. First, Cuff is required for localization of both Thoc5 and UAP56 to nuclear foci, the sites of nascent piRNA precursor transcription. Second, Cuff copurifies with Thoc5 from cellular extract, and this interaction is at least partially RNA-independent, indicating that Cuff can bind the TREX complex either directly or through other proteins. Importantly, a close paralog of Cuff, CG9125, does not form a complex with TREX, supporting the specificity of the interaction. Finally, tethering of Cuff to an artificial intronless reporter enhances binding of TREX to the reporter transcript. This experiment provides direct evidence that the presence of Cuff in the vicinity of a nascent transcript stimulates the association of TREX with the RNA. Together, these results suggest that Cuff and TREX form a complex on dual-strand piRNA loci, and Cuff enhances loading of TREX on nascent RNA (Hur, 2016).

Together with previously reported results, the current data can be integrated into a model that suggests that interaction with chromatin-bound Cuff helps to recruit TREX to dual-strand piRNA cluster loci in the nucleus to ensure its binding with nonspliced nascent piRNA transcripts. Previous studies showed that Cuff is enriched on chromatin of dual-strand piRNA clusters, likely through formation of the complex with Rhi that directly binds the histone H3K9me3. This study proposes that, similar to the result that were obtained using an artificial reporter, Cuff enhances loading of TREX on nascent piRNA precursors. Unfortunately, it is impossible to test the association of TREX with piRNA precursors in cuff mutants, as cuff deficiency leads to almost complete elimination of these RNAs. The current model proposes a novel splicing-independent mechanism of TREX loading in which a chromatin-binding protein (Rhi) and an adapter protein (Cuff) result in locus-specific, but sequence-independent, loading of an RNA-binding protein complex on nascent transcripts. It will be interesting to explore whether other proteins that associate with specific chromatin marks mediate loading of TREX on other intronless transcripts. It is hypothesized that chromatin structure and associated chromatin proteins might play crucial roles in guiding loading of other non-sequence-specific RNA-binding proteins on nascent RNA. Several RNA-binding proteins are cotranscriptionally loaded on nascent RNA and may even shuttle with processed mRNA to the cytoplasm. Therefore, the origin of an RNA in a particular chromatin environment might have long-lasting and far-reaching effects on its stability, subcellular localization, and translation efficiency. Quantitative profiling of the association between RNA-binding proteins and RNA originating from different chromatin environments will provide the ultimate test of this hypothesis (Hur, 2016).

TREX was proposed to play roles in post-transcriptional pre-mRNA processing and nuclear export of mRNA. However, the current results showed that only a small number of genes change their expression in TREX mutants. Similar results were previously reported using knockdown of TREX components in fly and mammalian cells, suggesting that the function of TREX in mRNA biogenesis in metazoa might be redundant with functions of other factors. Using two independent approaches, analysis of chromatin-associated nascent transcripts and in situ RNA hybridization, this study showed that TREX is required for the accumulation of nascent piRNA precursors at sites of transcription. These experiments cannot formally rule out the possibility that TREX stabilizes nascent piRNA transcripts and prevents their cotranscriptional degradation. However, the decrease in Pol II occupancy on piRNA clusters in the thoc5^e/1 mutant suggests that TREX might be required for efficient transcription of piRNA precursors. TREX has previously been shown to prevent formation of RNA–DNA hybrids (R loops) between the nascent transcript and the DNA template in mammalian cells (Dominguez-Sanchez, 2011), establishing a possible mechanism by which TREX could facilitate transcription. The impact of TREX on transcription does not eliminate the possibility that it also functions on later steps of piRNA biogenesis. Particularly, it will be important to determine whether TREX plays a role in the export of piRNA precursors from the nucleus to the cytoplasm, where final processing into mature piRNAs takes place (Hur, 2016).

p53 and PI3K/AKT signalings are up-regulated in flies with defects in the THO complex

The THO complex (THO) is an evolutionary conserved protein required for the formation of export-competent mRNP. The growing evidence indicates that the metazoan THO plays important roles in cell differentiation and cellular stress response. But the underlying mechanisms are poorly understood. This study examined the relevance of THO to cellular signaling pathways involved in cell differentiation and cellular stress response. When the endogenous p53 level was examined in the testis, it was found to be sustained much longer during spermatogenesis in the THO mutant compared to that of wild-type. In flies with impaired THO, overexpression of p53 by eye-specific GAL4 not only enhanced p53-mediated retinal degeneration, but p53 level was also elevated compared to the control flies. Since the body size of the THO mutant flies was significantly larger than control flies, whether the PI3K/AKT signaling is enhanced in the mutant flies was also examined. The results showed that the endogenous level of phosphorylated AKT, which is the active form, was highly elevated in the THO mutants. Taken together, these results suggested that both p53 and PI3K/AKT signalings are up-regulated in the flies with impaired THO (Moon, 2013).

A previous report showed the reduced life span and the increased susceptibility to the environmental stresses in mutant flies for Drosophila THO subunits (Kim, 2011). To understand the underlying mechanisms of these defects, this study investigated genetic interactions of THO with 2 cellular signaling pathways, p53 and PI3K/AKT pathways (Moon, 2013).

The following evidence suggests that defects in the function of THO cause up-regulation of p53 in a cell autonomous manner. First, endogenous level of p53 was sustained much longer during spermatogenesis in the male germline lacking THO compared to the control germline. FRT/FLP-based clonal analysis showed that p53 level was cell-autonomously sustained in the mutant clone. Second, mutations in the THO subunit genes elevated the level of overexpressed p53 in the eye, showing an increased sensitivity to p53-mediated apoptosis. Third, the sensitivity to p53-mediated apoptosis was directly correlated with the genetic background; the more severe defect in THO the genetic background had, the greater the sensitive to p53-mediated apoptosis was (Moon, 2013).

Why p53 is up-regulated in the flies with impaired THO? The fact that the nucleolar integrity was severely disrupted in flies lacking THO let to a postulate that disruption of nucleolus might be a good candidate to answer for this question (Moon, 2011). It has been known that disruption of nucleolus mediated stabilization of p53 in response to DNA damage and other stresses in mammalian cells. In addition, it has been reported that genetic disruption of nucleolus by knocking out murine TIF-1A gene caused p53 to be stabilized by dissociating it from MDM2. However, it is doubtful that the same is true in the Drosophila model. First, mutations in THO subunits caused nucleolar disruption only in certain types of cells including male germline and salivary gland cells (Moon, 2011). Moreover, this study failed to find any signs of nucleolar disruption in the THO-deficient eyes which were sensitized to p53-mediated apoptosis. Second, it has recently been reported that p53 level was not significantly increased in the eyes of viriato mutant in which nucleolar architecture was severely compromised (Marinho, 2011). Finally, the Drosophila homolog of MDM2, which plays a key role in nucleolar disruption-mediated p53 stabilization in mammalian cells, has not been found to date. Consistent with these facts, in Drosophila, it has been shown that posttranslational modification rather than abundance was sufficient to activate p53 signaling in response to DNA damage. For these reasons, it is speculated that nucleolar disruption is not directly involved in the up-regulation of p53 in the THO mutant flies. An alternative possibility is that the phenotype of condensed chromatin structure in thoc5 might represent genomic instability, and the genomic instability could lead to activation of MNK, Drosophila homolog of CHK2, which activates p53 following DNA damage. But the fact that DNA damage activates p53 without significant changes in protein level is inconsistent with the current findings which show obvious changes of p53 level in the testis. Another alternative explanation for these ambiguities is that upregulation of p53 in the THO mutant may be restricted to certain limited types of cells, and the mechanisms underlying this may also be different depending on cell types. To clarify these issues further studies are certainly required (Moon, 2013).

In addition to p53 signaling, PI3K/AKT signaling was examined in the THO mutant flies. It has been well established that PI3K/AKT signaling pathway is a key player in regulating life span as well as body size in Drosophila. Combined with the previously reported lifespan reduction (Kim, 2011), increased body size in the THO mutant flies compared to control is well matched with the known phenotypes of mutant flies with defects in PI3K/AKT signaling pathway. Although no global increase in PI3K/AKT signaling was detected in the THO mutant flies, a cell-autonomous elevation of endogenous p-AKT level in the mutant male germline provided a piece of evidence for the relevance of Drosophila THO with PI3K/AKT signaling (Moon, 2013).

Another interesting finding in this report is that the levels of both p53 and p-AKT are very high in the wild-type male germ germline. If p53 is important for spermatogenesis, why p53-null flies are not sterile? With regard to the female germline, it has recently been reported that DNA double strand breaks formed during meiotic recombination provoked activation of p53, and unrepaired DNA breaks during meiotic recombination led to sustained p53 activity. But it has been known that meiotic recombination is very rare in the Drosophila male germline, and this study showed that p53 was detected only in the pre-meiotic germline. Thus, it is unlikely that the role of p53 in male germline is similar to that in female germline. Certainly these issues will be a good topic for future study (Moon, 2013).

Taken together, this study found the significant genetic interactions of THO with 2 cellular signaling pathways, p53 and PI3K/AKT signaling pathways. Both signalings were up-regulated by THO dysfunction in a cell autonomous manner. However, it seems unlikely that THO generally plays a major role in regulating these signaling pathways, because not only Western blot analysis of whole-fly extract, but also FRT/FLP-mediated clonal analysis in the imaginal discs showed no significant changes in the endogenous levels of both p53 and p-AKT in the THO mutants. It seems rather likely that the effect of THO dysfunction on these two signaling pathways is different depending on cell types; it might be generally mild in most cells except certain types of cells such as germline (Moon, 2013).

The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. This study isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, this study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes (Moon, 2011).

During spermatogenesis, the coordinated action of many gene products is essential for proper differentiation of sperm. In Drosophila, ∼25% of all genes expressed in the testis are testis-specific or testis-enriched, and most of these genes are transcribed in primary spermatocytes and stored until needed. Testis-specific gene regulation programs might ensure the coordinated expression of this large number of genes from transcription to translation. In Drosophila, two distinct classes of meiotic arrest genes, aly-class and can-class genes, represent testis-specific transcription regulation modules. The can-class genes, cannonball (can), spermatocyte arrest (sa), meiosis I arrest (mia) and no hitter (nht), encode the testis-specific TBP-associated factors (tTAFs), suggesting that their products form a testis-specific TFIID complex in primary spermatocytes. Interestingly, tTAFs, together with Polycomb group (PcG) proteins, mainly localize to a subcompartment of the nucleolus, rather than to euchromatin. Lack of tTAFs not only disrupts the nucleolar localization of PcG proteins but also causes PC to accumulate at tTAF target promoters. These findings suggested that tTAFs might also antagonize the Polycomb repressor complex (PRC1) to control the coordinated transcription of target genes (Moon, 2011).

Most aly-class gene products form tMAC, a testis-specific meiotic arrest complex paralogous to Myb-MuvB. The aly-class gene products are mainly localized to euchromatin in primary spermatocytes, and this localization is essential for their function, suggesting that the major role of the tMAC complex is transcriptional activation of testis-specific genes. Thus, it now seems evident that the coordinated transcription of testis-specific genes is regulated by two specific complexes: tTAFs and tMAC. However, little is known about post-transcriptional regulation in the testis. This study shows that the THO complex, an evolutionarily conserved complex involved in the co-transcriptional formation of export-competent messenger ribonucleoproteins (mRNPs), is a novel regulator of Drosophila spermatogenesis (Moon, 2011).

The THO complex was first found in budding yeast, Saccharomyces cerevisiae, as a multisubunit protein complex composed of Hpr1, Tho2, Mft1 and Thp2. Yeast cells that lack the THO complex show transcription impairment and transcription-dependent hyper-recombination phenotypes, implying that the THO complex connects transcription elongation to mitotic recombination. Subsequent studies showed that the THO complex, together with the mRNA export adaptor proteins Yra1 and Sub2, forms a larger complex called TREX (transcription-export complex), which is required for the co-transcriptional export of bulk mRNAs. Metazoans also have a functional homolog of the THO complex, but its subunit composition and function are slightly different from those of budding yeast (Masuda, 2005; Rehwinkel, 2004). The metazoan THO complex lacks Mft1 and Thp2, but contains three other subunits, THOC5, THOC6 and THOC7, instead. Unlike budding yeast, metazoan cells require the THO complex for nuclear export of only a subset of transcripts, but almost nothing is known about the common features of the target transcripts. Although it is now clear that, at the single-cell level, a role of the THO complex in mRNP biosynthesis is conserved throughout evolution from yeast to human (for a review, see Jimeno and Aguilera, 2010), its exact role in various types of cells at the multicellular organism level is still largely unknown. Recent studies using conditional knockout or a hypomorphic mutant mouse model have provided evidence that the THO complex has specific roles in cell differentiation during development. It has also recently been reported that the Drosophila THO complex is required for normal development through collaboration with E(Y)2 (or ENY2), a multifunctional protein important for transcription activation and mRNA export (Kopytova, 2010; Moon, 2011 and references therein).

This study reports that a novel meiotic arrest gene, garmcho (garm), encodes the Drosophila THOC5 homolog. Flies lacking THOC5 showed complete male sterility with a meiotic arrest phenotype. Interestingly, unlike any other known meiotic arrest mutants, the nucleolar structure was severely disrupted in garm mutant primary spermatocytes. Both tTAF and PC proteins were abnormally distributed, whereas the expression and nuclear export of the three tTAF target mRNAs examined were mainly unaffected in mutant primary spermatocytes. Taken together, these data provide additional evidence that the THO complex is involved in a specific cell differentiation program, Drosophila spermatogenesis, probably by participating in the establishment or maintenance of nucleolar integrity in spermatocytes (Moon, 2011).

The phenotypes of garm are different from those of other meiotic arrest mutants. First, the chromosomes were more condensed in arrested mutant spermatocytes than in wild-type spermatocytes. In other known meiotic arrest mutants, spermatocytes have chromosomes either similar to or less condensed than wild type. Second, unlike other mutants, the nucleoli were severely disrupted in garm mutant primary spermatocytes. Taken together, these findings suggest that garm represents a novel class of meiotic arrest gene, and that its gene product might play a role distinct from that of other meiotic arrest gene products, which are involved in the transcription of target genes. Indeed, THOC5 (encoded by garm) is a subunit of the THO complex, which is an evolutionarily conserved protein complex required for mRNP biosynthesis. So far, garm (thoc5) is the only meiotic arrest gene whose gene product is likely to be involved in a post-transcriptional step (Moon, 2011).

THOC5 is likely to function in spermatocytes as a subunit of the THO complex. First, all subunits of the THO complex examined were not only colocalized in spermatocytes but also co-immunoprecipitated with each other, suggesting that they form a stable complex in spermatocytes. Second, in mutant spermatocytes lacking one of the THO subunits, not only was the localization of other THO subunits disrupted, but also their expression level was significantly reduce. Third, hypomorphic mutants of both thoc6 and thoc7 appeared to show mild disruption of the nucleoli, although they did not show the typical meiotic arrest phenotype (Moon, 2011).

What is the specific role of the THO complex in Drosophila spermatogenesis? The finding that the nucleolar structure was severely disrupted in thoc5 mutants suggests that the THO complex might be required for the proper organization of the nucleolus. In accordance with this, all subunits of the THO complex accumulate at the peri-nucleolar region in pre-meiotic spermatocytes. Recent progress in understanding of the nucleolus suggests that not only does it function as the ribosome-producing factory, but it also regulates mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles. Consistent with these non-traditional functions, many proteins unrelated to ribosome assembly are found in the nucleolus. Moreover, the integration of many different sources of protein-protein interaction data showed that the spliceosomal complex is one of the major protein complexes in human nucleolus, suggesting that the spliceosomes are structural components of the nucleolus. Thus, it is speculated that the nucleolar disruption found in thoc5 spermatocytes might be caused by malformation of the spliceosomal complex, because the THO complex associates with splicesomal proteins independently of transcription (Cheng, 2006; Masuda, 2005). One of the common phenotypes caused by nucleolar disruption in mammalian cells is the p53-mediated cellular stress response, which includes cell-cycle arrest and apoptosis. To test whether the meiotic arrest phenotype seen in this study was also mediated by p53, genetic interactions were examined between thoc5 and p53. However, lack of p53 failed to suppress the meiotic arrest phenotype caused by thoc5 , suggesting that p53 is not required for the meiotic arrest phenotype seen in thoc5 mutant spermatocytes. This also suggested, if nucleolar disruption was the cause of the meiotic arrest, that some other unknown pathways are required for cell-cycle arrest in Drosophila spermatocyte. Alternatively, the THO complex might simply be required for the nuclear export of mRNAs for meiotic cell-cycle regulators, such as CycB or twine (Moon, 2011).

The finding that PC localization to the nucleolus requires tTAFs raised a hypothesis that the nucleolus acts as a sequestering compartment for counteracting transcriptional silencing by PcG proteins in Drosophila spermatocytes. If this is the case, transcription of tTAF target genes might be affected in thoc5 mutant spermatocytes because the nucleolar localizations of both PC and the tTAF SA are abnormal in the thoc5 mutant. Surprisingly, however, tTAF target genes were transcribed; and even more surprisingly, their transcripts were still exported to cytoplasm in thoc5 mutant spermatocytes, suggesting that the nucleolar localization of tTAFs and PcG proteins is not essential for counteracting transcriptional silencing by PcG proteins. In addition to this, a male-sterile gene, bol, which is a target of another testis-specific meiotic arrest complex, tMAC, was also expressed and exported independently of THOC5. Taken together, these results suggest that the meiotic arrest phenotype in thoc5 might not be caused by the failure of the canonical THO function, mRNA export (Moon, 2011).

The finding that don juan (dj) transcript normally appeared to be retained in the nucleus at the pre-meiotic stage of spermatogenesis is very interesting. The transcription of dj mRNA is known to be initiated in early spermatogenesis, but its translation is normally delayed until meiotic divisions are completed (Santel, 1997). Although it has been reported that translational repression of dj mRNA is mediated by the TRE (dj translational repression element) located at the 5'-UTR (Blumer, 2002), it is speculated that nuclear retention of dj mRNA might be an additional mechanism by which dj mRNA is translationally suppressed. In mammalian cells, a novel regulation mechanism of gene expression through RNA nuclear retention has been recently proposed. In this model, certain mRNAs containing elements for adenosine-to-inosine editing within their 3'-UTR are retained in nuclear paraspeckles, and may be released when the demand for their protein products increases. Although it is unclear whether Drosophila has a similar regulatory mechanism, the nuclear retention of dj mRNA in pre-meiotic spermatocytes might be a sign of the existence of such a mechanism. Further studies are required to clarify this issue. From the finding that nuclear mRNA retention is not generally applicable to other tTAF target genes, the possibility that the dj probe might cross-hybridize with some other nuclear RNAs concentrated in a certain nuclear structure, such as the Y-loop, cannot be ruled out. However, this is unlikely because the signal was absent in mia mutant spermatocytes in which dj is not expressed; moreover, the nuclear dj message was still detectable in the testes of XO males, which lack the Y-loop. A previous report (Santel, 1997) that showed a strong cytoplasmic signal of dj mRNA by whole-mount in situ hybridization with colorimetric detection is also inconsistent with the current result. However, this study also failed to detect a clear signal of dj massages in the nucleus by a similar method. Thus, the discrepancy was probably due to methodological differences. Whether the signal detected by a dj anti-sense probe represents genuine dj message or not, nuclear retention of this RNA appears to be dependent on the THO complex (Moon, 2011).

The THO complex is not a testis-specific protein complex. Why, then, might the testis be more sensitive to loss of THOC5 than other tissues are? There are possible reasons for this. First, THOC5 might not be essential for the function of the THO complex. In the thoc5 mutant, a significant amount of HPR1 was still detected in the nucleus, although its level was greatly reduced. Interestingly, the residual HPR1 was mainly located near the chromatins rather than the nucleolus. Thus, the residual subunits might still have some degree of activity, and this residual activity might be sufficient for most cells, but not enough for other cells, such as spermatocytes. Second, in addition to mRNP biogenesis, the THO complex might have a non-canonical function in the spermatocytes, such as the organization and/or maintenance of nucleolar structure, and possibly the localization of some nuclear proteins and RNAs. Third, spermatogenesis requires the coordinated expression of a large number of genes. To ensure that the spermatocytes regulate gene expression in a coordinated manner from transcription to nuclear export, the THO complex might still have a role, although its specific target mRNA was not detected (Moon, 2011).

In summary, disruption of THOC5 caused severe defects in the primary spermatocyte nucleus, including nucleolar disruption, abnormal distribution of proteins (SA and PC) and an RNA transcript (dj), suggesting that it has a role in the establishment or maintenance of subnuclear structure in Drosophila primary spermatocytes. The main causative factor of meiotic arrest in thoc5 mutants might be the disruption of subnuclear structure rather than the defect in nuclear mRNA export (Moon, 2011).

In addition to the meiotic arrest phenotype reported in this study, the thoc5 mutant also has other phenotypes, including wing bubble and uncoordinated behavior. Recent work also showed that longevity and tolerance to environmental stress were significantly reduced in the THO mutants. This suggests that the role of the THO complex is not limited to spermatogenesis, but is also important for other types of cells. When this study examined whether a similar nucleolar defect is seen in other cell types, no similar defect was found in other cell types except the salivary gland cells. This suggests that the specific role of the THO complex is different depending on the cell type. It will be interesting to clarify the specific roles of the THO complex in other types of cells (Moon, 2011).

Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. This study reports that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster (Rehwinkel, 2004).

Search PubMed for articles about Drosophila Thoc5

Aguilera, A. (2005). mRNA processing and genomic instability. Nat Struct Mol Biol 12: 737-738. PubMed ID: 16142225

Blumer, N., Schreiter, K., Hempel, L., Santel, A., Hollmann, M., Schafer, M. A. and Renkawitz-Pohl, R. (2002). A new translational repression element and unusual transcriptional control regulate expression of don juan during Drosophila spermatogenesis. Mech Dev 110: 97-112. PubMed ID: 11744372

Cheng, H., Dufu, K., Lee, C. S., Hsu, J. L., Dias, A. and Reed, R. (2006). Human mRNA export machinery recruited to the 5' end of mRNA. Cell 127: 1389-1400. PubMed ID: 17190602

Chi, B., Wang, Q., Wu, G., Tan, M., Wang, L., Shi, M., Chang, X. and Cheng, H. (2013). Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA. Nucleic Acids Res 41: 1294-1306. PubMed ID: 23222130

Chi, B., Wang, K., Du, Y., Gui, B., Chang, X., Wang, L., Fan, J., Chen, S., Wu, X., Li, G. and Cheng, H. (2014). A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18. Nucleic Acids Res 42: 7305-7318. PubMed ID: 24782531

Czech, B., Preall, J. B., McGinn, J. and Hannon, G. J. (2013). A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway. Mol Cell 50: 749-761. PubMed ID: 23665227

Dominguez-Sanchez, M. S., Barroso, S., Gomez-Gonzalez, B., Luna, R. and Aguilera, A. (2011). Genome instability and transcription elongation impairment in human cells depleted of THO/TREX. PLoS Genet 7: e1002386. PubMed ID: 22144908

Gatfield, D., Le Hir, H., Schmitt, C., Braun, I. C., Kocher, T., Wilm, M. and Izaurralde, E. (2001). The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr Biol 11: 1716-1721. PubMed ID: 11696332

Gatfield, D. and Izaurralde, E. (2002). REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export. J Cell Biol 159: 579-588. PubMed ID: 12438415

Guria, A., Tran, D. D., Ramachandran, S., Koch, A., El Bounkari, O., Dutta, P., Hauser, H. and Tamura, T. (2011). Identification of mRNAs that are spliced but not exported to the cytoplasm in the absence of THOC5 in mouse embryo fibroblasts. RNA 17: 1048-1056. PubMed ID: 21525145

Hur, J.K., Luo, Y., Moon, S., Ninova, M., Marinov, G.K., Chung, Y.D. and Aravin, A.A. (2016). Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila. Genes Dev 30: 840-855. PubMed ID: 27036967

Jimeno, S. and Aguilera, A. (2010). The THO complex as a key mRNP biogenesis factor in development and cell differentiation. J Biol 9: 6. PubMed ID: 20236444

Katahira, J. (2012). mRNA export and the TREX complex. Biochim Biophys Acta 1819: 507-513. PubMed ID: 22178508

Kim, N. and Jinks-Robertson, S. (2011). Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast. DNA Repair (Amst) 10: 953-960. PubMed ID: 21813340

Kopytova, D. V., Krasnov, A. N., Orlova, A. V., Gurskiy, D. Y., Nabirochkina, E. N., Georgieva, S. G. and Shidlovskii, Y. V. (2010). ENY2: couple, triple...more? Cell Cycle 9: 479-481. PubMed ID: 20090412

Kopytova, D. V., Orlova, A. V., Krasnov, A. N., Gurskiy, D. Y., Nikolenko, J. V., Nabirochkina, E. N., Shidlovskii, Y. V. and Georgieva, S. G. (2010). Multifunctional factor ENY2 is associated with the THO complex and promotes its recruitment onto nascent mRNA. Genes Dev 24: 86-96. PubMed ID: 20048002

Le Thomas, A., Toth, K. F. and Aravin, A. A. (2014). To be or not to be a piRNA: genomic origin and processing of piRNAs. Genome Biol 15: 204. PubMed ID: 24467990

Marinho, J., Casares, F. and Pereira, P. S. (2011). The Drosophila Nol12 homologue viriato is a dMyc target that regulates nucleolar architecture and is required for dMyc-stimulated cell growth. Development 138: 349-357. PubMed ID: 21177347

Masuda, S., Das, R., Cheng, H., Hurt, E., Dorman, N. and Reed, R. (2005). Recruitment of the human TREX complex to mRNA during splicing. Genes Dev 19: 1512-1517. PubMed ID: 15998806

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157: 1364-1379. PubMed ID: 24906153

Moon, S., Cho, B., Min, S. H., Lee, D. and Chung, Y. D. (2011). The THO complex is required for nucleolar integrity in Drosophila spermatocytes. Development 138: 3835-3845. PubMed ID: 21828100

Moon, S. and Chung, Y. D. (2013). p53 and PI3K/AKT signalings are up-regulated in flies with defects in the THO complex. Mol Cells 35: 261-268. PubMed ID: 23475424

Nojima, T., Hirose, T., Kimura, H. and Hagiwara, M. (2007). The interaction between cap-binding complex and RNA export factor is required for intronless mRNA export. J Biol Chem 282: 15645-15651. PubMed ID: 17363367

Rehwinkel, J., Herold, A., Gari, K., Kocher, T., Rode, M., Ciccarelli, F. L., Wilm, M. and Izaurralde, E. (2004). Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster. Nat Struct Mol Biol 11: 558-566. PubMed ID: 15133499

Santel, A., Winhauer, T., Blumer, N. and Renkawitz-Pohl, R. (1997). The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif. Mech Dev 64: 19-30. PubMed ID: 9232593

Zhang, F., Wang, J., Xu, J., Zhang, Z., Koppetsch, B. S., Schultz, N., Vreven, T., Meignin, C., Davis, I., Zamore, P. D., Weng, Z. and Theurkauf, W. E. (2012). UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery. Cell 151: 871-884. PubMed ID: 23141543

Zhang, Z., Wang, J., Schultz, N., Zhang, F., Parhad, S. S., Tu, S., Vreven, T., Zamore, P. D., Weng, Z. and Theurkauf, W. E. (2014). The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing. Cell 157: 1353-1363. PubMed ID: 24906152

Zhou, Z., Luo, M. J., Straesser, K., Katahira, J., Hurt, E. and Reed, R. (2000). The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407: 401-405. PubMed ID: 11014198

date revised: 20 April 2016

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