InteractiveFly: GeneBrief

twin: Biological Overview | References

Gene name - twin

Synonyms - CCR4

Cytological map position - 95F1-95F2

Function - enzyme

Keywords - mRNA deadenylation, regulates cyclin poly(A) tail length, microRNA pathway, deadenylation complex

Symbol - twin

FlyBase ID: FBgn0039168

Genetic map position - 3R: 20,023,310..20,047,687 [-]

Classification - CCR4 mRNA deadenylase exonuclease subunit, LRR protein

Cellular location - cytoplasmic

NCBI link: EntrezGene

Twin orthologs: Biolitmine

Recent literature
Fu, Z., Geng, C., Wang, H., Yang, Z., Weng, C., Li, H., Deng, L., Liu, L., Liu, N., Ni, J. and Xie, T. (2015). Twin promotes the maintenance and differentiation of germline stem cell lineage through modulation of multiple pathways. Cell Rep 13: 1366-1379. PubMed ID: 26549449
The central question in stem cell regulation is how the balance between self-renewal and differentiation is controlled at the molecular level. This study uses germline stem cells (GSCs) in the Drosophila ovary to demonstrate that the Drosophila CCR4 homolog Twin is required intrinsically to promote both GSC self-renewal and progeny differentiation. Twin/CCR4 is one of the two catalytic subunits in the highly conserved CCR4-NOT mRNA deadenylase complex. Twin works within the CCR4-NOT complex to intrinsically maintain GSC self-renewal, at least partly by sustaining E-cadherin-mediated GSC-niche interaction and preventing transposable element-induced DNA damage. It promotes GSC progeny differentiation by forming protein complexes with differentiation factors Bam and Bgcn independently of other CCR4-NOT components. Interestingly, Bam can competitively inhibit the association of Twin with Pop2 in the CCR4-NOT complex. Therefore, this study demonstrates that Twin has important intrinsic roles in promoting GSC self-renewal and progeny differentiation by functioning in different protein complexes.

Niinuma, S., Fukaya, T. and Tomari, Y. (2016). CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence. RNA. PubMed ID: 27484313
MicroRNAs (miRNAs) recruit the CCR4-NOT complex, which contains two deadenylases, CCR4 and CAF1, to promote shortening of the poly(A) tail. Although both CCR4 and CAF1 generally have a strong preference for poly(A) RNA substrates, it has been reported from yeast to humans that they can also remove non-A residues in vitro to various degrees. However, it remains unknown how CCR4 and CAF1 remove non-A sequences. This study shows that Drosophila miRNAs can promote the removal of 3'-terminal non-A residues in an exonucleolytic manner, but only if an upstream poly(A) sequence exists. This non-A removing reaction is directly catalyzed by both CCR4 and CAF1 and depends on the balance between the length of the internal poly(A) sequence and that of the downstream non-A sequence. These results suggest that the CCR4-NOT complex has an intrinsic activity to remove the 3'-terminal non-A modifications downstream from the poly(A) tail
Meers, M. P., Henriques, T., Lavender, C. A., McKay, D. J., Strahl, B. D., Duronio, R. J., Adelman, K. and Matera, A. G. (2017). Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity. Elife 6. PubMed ID: 28346137
Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, a 'histone replacement' system was used in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. Global dysregulation of mRNA levels was observed in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, it was found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. A post-transcriptional role is proposed for modification of replication-dependent H3K36 in the control of metazoan gene expression.
Sgromo, A., Raisch, T., Backhaus, C., Keskeny, C., Alva, V., Weichenrieder, O. and Izaurralde, E. (2017). Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets. RNA [Epub ahead of print]. PubMed ID: 29255063
Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. This study investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, it was found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an alpha-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. It was further shown that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40.
Kordyukova, M., Sokolova, O., Morgunova, V., Ryazansky, S., Akulenko, N., Glukhov, S. and Kalmykova, A. (2019). Nuclear Ccr4-Not mediates the degradation of telomeric and transposon transcripts at chromatin in the Drosophila germline. Nucleic Acids Res. PubMed ID: 31724732
Ccr4-Not is a highly conserved complex involved in cotranscriptional RNA surveillance pathways in yeast. In Drosophila, Ccr4-Not is linked to the translational repression of miRNA targets and the posttranscriptional control of maternal mRNAs during oogenesis and embryonic development. This study describes a new role for the Ccr4-Not complex in nuclear RNA metabolism in the Drosophila germline. Ccr4 depletion results in the accumulation of transposable and telomeric repeat transcripts in the fraction of chromatin-associated RNA; however, it does not affect small RNA levels or the heterochromatin state of the target loci. Nuclear targets of Ccr4 mainly comprise active full-length transposable elements (TEs) and telomeric and subtelomeric repeats. Moreover, Ccr4-Not foci localize at telomeres in a Piwi-dependent manner, suggesting a functional relationship between these pathways. Indeed, this study detected interactions between the components of the Ccr4-Not complex and piRNA machinery, which indicates that these pathways cooperate in the nucleus to recognize and degrade TE transcripts at transcription sites. These data reveal a new layer of transposon control in the germline, which is critical for the maintenance of genome integrity.
Morgunova, V., Kordyukova, M., Mikhaleva, E. A., Butenko, I., Pobeguts, O. V. and Kalmykova, A. (2021). Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development. PLoS One 16(10): e0258156. PubMed ID: 34624021
Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. The mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila was studied. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. This study found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction (Morgunona, 2021).


The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. Homologs for almost all subunits of this complex have been identified in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1 (FlyBase name: Pop2), were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins are ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells leads to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length is also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and that it plays a role in general and regulated mRNA deadenylation (Temme, 2004).

A characteristic feature of mRNA is its rapid turnover, permitting protein production to be continuously adjusted to the physiological requirements. Each mRNA has a characteristic half-life; within a given cell, the half-lives of different mRNAs differ by more than a factor of 10. A short half-life leads to a rapid adjustment of the steady-state level of an mRNA upon either an up- or downregulation of the rate of transcription. The half-lives of some mRNAs can also be regulated. For example, pronounced instability of the mRNA encoding the Hsp70 heat shock protein contributes, in addition to transcriptional repression, to its very low steady-state level at normal temperatures, and stabilization of the message plays a role in Hsp70 induction upon heat shock (Temme, 2004).

Two general pathways of mRNA decay have been characterized in yeast. Exonucleolytic removal of the poly(A) tail (deadenylation) is the first step in both, and this can also be the rate-limiting and regulated step. In the major decay pathway, the second step is the hydrolytic cleavage of the m7GpppN cap, which occurs only after the poly(A) tail has been shortened to about 10 nt. The RNA is then degraded from its 5' end. In the minor pathway, the step following deadenylation consists of 3' exonucleolytic degradation of the mRNA body. In yeast, two different enzyme complexes are involved in deadenylation. The more important one contains Ccr4p as the major catalytic subunit (Tucker, 2001; Tucker, 2002; Chen, 2002). A second subunit, Pop2p/Caf1p, is a member of the RNase D family of 3' exonucleases and also has 3' exonuclease activity (Daugeron, 2001; Thore, 2003), but under normal conditions the activity of Ccr4p dominates (Chen, 2002; Tucker, 2002). Additional polypeptides in the complex are Not1-5p, Caf40p and Caf130p (Chen, 2001). ccr4δ and pop2δ/caf1δ mutants grow slowly and display a reduction in the rate of mRNA deadenylation (Tucker, 2001). not2δ and not5δ also affect mRNA deadenylation, albeit more weakly than ccr4 and pop2/caf1 mutations (Tucker, 2002). The CCR4-NOT complex is also believed to play a role in transcriptional regulation (Collart, 2003; Denis, 2003). The second deadenylase of Saccharomyces cerevisiae is composed of the Pan2 and Pan3 proteins (Boeck, 1996; Brown, 1996). Deletion mutants show an increase in the steady-state length of poly(A) tails, and the Pan2p/Pan3p complex catalyzes the residual deadenylation observed in ccr4 or pop2/caf1 mutants (Tucker, 2001). The Pan2p/Pan3p complex (Brown, 1998) appears to be involved in an initial shortening of overly long poly(A) tails to a length of 50-90 nt (Temme, 2004).

In metazoans, like in yeast, deadenylation is the first and often rate-limiting step of mRNA decay. For example, the unstable Hsp70 mRNA mentioned above is rapidly deadenylated at normal growth temperature; retarded deadenylation during heat shock is at least partially responsible for stabilization of the message. Subsequent decay steps proceed rapidly and without detectable intermediates in vivo. However, it is generally accepted that both pathways of mRNA decay described above also exist in animal cells (Couttet, 1997; Decker, 2002; van Hoof, 2002). The situation is more complex with respect to deadenylating enzymes. The CCR4-NOT complex is conserved in man and other species, but the human genome contains two homologs of yeast POP2/CAF1 (Albert, 2000), and metazoans have four different homologs of the yeast CCR4 gene (Dupressoir, 2001). Two of these, Xenopus Nocturnin and human CCR4, have been demonstrated to have poly(A)-degrading activity in vitro (Chen, 2002; Baggs, 2003). Thus, there may be different versions of the CCR4-NOT complex in higher eukaryotes. In human cells, the Pan2/Pan3 complex has also been characterized (Uchida, 2004). Finally, a third enzyme, the poly(A)-specific ribonuclease (PARN; initially called DAN), has been identified in vertebrates and is thought to be responsible for the so-called default deadenylation during Xenopus oocyte maturation (Körner, 1998; Wickens, 2000), a developmentally controlled general poly(A) tail degradation serving in translational silencing of mRNAs (Temme, 2004).

The CCR4-NOT complex is conserved in Drosophila. Homologs for most subunits of the yeast complex were found in the Drosophila genome (Denis, 2003). Gene references in FlyBase to the proteins in the complex are as follows: CCR4 (CG31137), CAF1 (Pop2), NOT1 (CG1884), NOT2 (CG2138), NOT3/5 (CG8426), NOT4 (CG31716) and CAF40 (CG14213). The Caf130 protein of yeast does not have any obvious counterpart in the fly, and such a protein also does not appear to be encoded in the human or other completely sequenced genomesDenis and Chen, 2003). The Not3 and Not5 proteins of yeast have sequence similarity to each other and are represented by a single protein in Drosophila as is the case in humans (Albert, 2000). Most importantly, the two catalytic subunits, CCR4 and CAF1, are conserved in Drosophila. In several different chromatographic separations, these two proteins were found to be associated with each other. However, sometimes a partial displacement of the CAF1 peak was seen with respect to the CCR4 peak, suggesting that the association may not be extremely tight. The nuclease activity associated with the two proteins was classified as a 3' exonuclease based on (1) the linear accumulation of acid-soluble product, (2) gradual shortening of a 5'-labeled RNA and (3) protection of the RNA by modification of the 3' end. The nuclease is likely to be poly(A) specific since degradation of a polyadenylated RNA resulted in the accumulation of a deadenylated product. A more thorough characterization will be meaningful only after further purification of the enzyme (Temme, 2004).

The composition of the CCR4-NOT complex is not entirely clear. A complex containing Ccr4p, Caf1p, Not1-5p, Caf40p and Caf130p has been purified from yeast (Chen, 2001). In contrast, TAP-tag purifications consistently revealed co-purification of Ccr4p, Not1p, Caf1p, Caf40p and Caf130p, but there was little evidence for an association of these proteins with Not2-5p. Similarly, purification of TAP-tagged human NOT2 resulted in co-purification of NOT1, both human homologs of POP2/CAF1, CAF40 and CCR4, but not of NOT3 and NOT4. The possibility has been discussed that the Not2-5 proteins may play distinct roles as opposed to Caf1p and Ccr4p. However, in S. cerevisiae, not2δ and not5δ mutations cause a significant decrease in the rate of poly(A) tail shortening, and not3δ and not4δ mutants also have slight defects (Tucker, 2002). These observations are consistent with an involvement of the entire complex in mRNA deadenylation. The data from Drosophila also support this idea: All subunits tested except CAF40 were found to be involved in deadenylation either by the analysis of mutant flies or in RNAi knock-down experiments. While knock-down of CAF40 had no effect on steady-state poly(A) length, this result does not rule out an involvement of the protein in deadenylation, since the success of RNAi could not be checked due to a lack of suitable antibodies (Temme, 2004).

Both ccr4δ and pop2/caf1δ mutants of S. cerevisiae have pronounced growth defects, at least in some genetic backgrounds, but they are viable. In contrast, NOT1 is an essential gene, and some combinations of mutations in subunits of the CCR4-NOT complex are synthetically lethal (Collart, 2003). The ccr4 allele of Drosophila described in this study is also viable. Although the level of the CCR4 protein is strongly reduced, the mutation is not detrimental to Drosophila development and the defect in deadenylation is relatively weak. An RNAi knock-down of CCR4 in S2 cells had essentially no detectable consequences for steady-state poly(A) tail length or the rate of Hsp70 deadenylation. While an incomplete depletion of the protein may contribute to the weakness of the knock-down phenotype, a more fundamental explanation for the relatively weak effect of CCR4 mutation or depletion may be the existence of other CCR4-like proteins. At least one of them, Nocturnin, is expected to contribute to mRNA deadenylation (Baggs, 2003), but the other two CCR4 paralogs might have a similar role. Interestingly, this functional redundancy does not occur in the female germ line; decreasing the level of CCR4 leads to female sterility (S Zaessinger and M Simonelig, unpublished data cited Temme, 2004). Biochemical fractionation experiments have also provided evidence for at least one additional poly(A)-degrading enzyme in S2 cells: Under slightly different conditions, a distinct poly(A)-specific 3' exonuclease has been separated from the one associated with CCR4 and CAF1. This nuclease and the one present in the flow-through of the DEAE column remain to be identified (Temme, 2004).

Immunostaining detected the CCR4 and CAF1 proteins in distinct cytoplasmic foci. Similar foci containing the mRNA decapping and degradation enzymes have been described in mammalian cells and in yeast and are thought to be the sites of mRNA decapping and decay. The mammalian foci also contain the CCR4 protein (Cougot, 2004), whereas an association of yeast Ccr4p with cytoplasmic foci is uncertain. The Drosophila CCR4 and CAF1 foci probably correspond to these cytoplasmic sites of mRNA decay found in other species. Decapping and degradation of the mRNA body are rapid events following complete deadenylation, and the mRNA's association with the proteins required for decapping appears to be accompanied (or even caused) by a loss of translation factors like eIF4E, eIF4G and Pab1p (Tharun, 2001). Thus, it is not hard to imagine that this mRNP rearrangement also involves the association with a particular cytoplasmic structure. Deadenylation, in contrast, is a continuous process during the mRNA's cytoplasmic lifetime. Thus, deadenylation presumably occurs while the RNA is being translated and would not be expected to be localized. In agreement with this, a substantial proportion of CCR4 seems to be widespread in the cytoplasm. Further experiments will be necessary to examine the functional significance of the presence of CCR4 and CAF1 in foci (Temme, 2004).

The CCR4-NOT complex of Drosophila is involved both in basal and regulated deadenylation. Deadenylation of the Hsp70 mRNA is regulated in two ways: First, any Hsp70 RNA made at normal growth temperature is deadenylated and degraded very rapidly. Although sequence dependence of deadenylation has not been directly examined, rapid decay of the Hsp70 message depends on the 3' UTR. Presumably, 3' UTR sequences activate the CCR4-NOT complex directly or indirectly via bound proteins. Second, heat shock leads to a stabilization of the message, apparently partly due to a reduced rate of deadenylation. As other rapidly deadenylated, unstable mRNAs are also stabilized during heat shock, it is possible that the deadenylating enzyme itself is a target of regulation rather than mRNA-specific factors. In agreement with this idea, growth conditions regulate a number of phosphorylation events in the yeast CCR4-NOT complex, some of proven physiological significance (Liu, 1997; Moriya, 2001; Lenssen, 2002). The double-stranded RNA-dependent protein kinase has been implicated in heat-shock regulation of mRNA stability in mammalian cells (Zhao, 2002), but the relevant target is unknown. The ubiquitin system has also been implicated in the regulation of mRNA stability during heat shock (Laroia, 1999). A further biochemical characterization of the CCR4-NOT complex will be essential in order to find out if and how this enzyme is regulated by heat shock (Temme, 2004).

LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

Post-transcriptional regulation of gene expression plays an essential role during oocyte maturation. This study reports that Drosophila MARF1 (Meiosis Regulator And mRNA Stability Factor 1), which consists of one RNA-recognition motif and six tandem LOTUS domains with unknown molecular function, is essential for oocyte maturation. When tethered to a reporter mRNA, MARF1 post-transcriptionally silences reporter expression by shortening reporter mRNA poly-A tail length and thereby reducing reporter protein level. This activity is mediated by the MARF1 LOTUS domain, which binds the CCR4-NOT deadenylase complex. MARF1 binds cyclin A mRNA and shortens its poly-A tail to reduce Cyclin A protein level during oocyte maturation. This study identifies MARF1 as a regulator in oocyte maturation and defines the conserved LOTUS domain as a post-transcriptional effector domain that recruits CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress their translation (Zhu, 2018).

RNA-binding proteins (RBPs) mediate post-transcriptional gene regulation by determining molecular fates of target RNAs. In addition to RNA-binding domains, RBPs often have additional auxiliary domains. These auxiliary domains may function as effector domains for post-transcriptional gene regulation directly through enzymatic activity or indirectly by mediating protein-protein interaction. Identifying these effector domains and their molecular functions is critical to understand the roles of RBPs in post-transcriptional gene regulatory mechanism (Zhu, 2018).

MARF1 is an RBP consisting of one RNA-recognition motif (RRM) followed by several tandem LOTUS domains (Limkain, Oskar, and Tudor containing proteins 5 and 7. Also called OST-HTH). Previous studies showed that mouse MARF1 is required for completion of meiosis in oogenesis by reducing protein and mRNA levels of retrotransposons and a few endogenous genes. However, the molecular mechanism by which MARF1 regulates gene expression remains unclear (Zhu, 2018).

LOTUS domains are conserved in bacteria, fungi, plants, and animals. In animals, LOTUS domain proteins are expressed almost exclusively in the germline and are implicated in RNA regulation. In Drosophila, these LOTUS domain proteins include Oskar, Tejas (human TDRD5), Tapas (human TDRD7), and MARF1 (Meiosis Regulator And mRNA Stability Factor 1 = GC17018. Human MARF1). However, the molecular function of the conserved LOTUS domain is not fully understood (Zhu, 2018).

This work studied the biological and molecular functions of Drosophila MARF1 and its LOTUS domains. MARF1 is essential for proper oocyte maturation by regulating cyclin protein levels. When tethered to a reporter mRNA, MARF1 caused shortening of reporter mRNA poly-A tail and reduced reporter protein level. This activity was mediated by MARF1 LOTUS domain. Consistent with this finding, it was found that MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain. Furthermore, it was found that MARF1 binds cyclin A mRNA, shortens its poly-A tail, and reduces Cyclin A protein level during oocyte maturation. Thus, this study uncovered the biological and molecular functions of Drosophila MARF1 and defined its conserved LOTUS domains as a post-transcriptional effector domain to recruit the CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress translation of the mRNAs (Zhu, 2018).

MARF1 is expressed in late-stage oocytes and is required for proper oocyte maturation by regulating cyclin protein levels. MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain to shorten target mRNA poly-A tails and thus reducing cyclin protein levels without changing cyclin mRNA levels. Thus, MARF1 LOTUS domain is defined as a post-transcriptional effector domain that binds the CCR4-NOT deadenylase complex (Zhu, 2018).

Recent studies by others showed that the LOTUS domains of Drosophila Oskar, Tejas, and Tapas bind germline DEAD-box RNA helicase Vasa to stimulate Vasa ATPase and helicase activities. Crystallographic studies showed that the LOTUS domain of Oskar forms a homodimer and that each of the monomer subunits binds the C-terminal domain of the Vasa DEAD-box helicase core on the side opposite to the dimerization interface. In contrast, this study shows that the MARF1 LOTUS domain binds the CCR4-NOT deadenylase complex, but does not bind Vasa, Oskar, or another molecule of MARF1 (Zhu, 2018).

The LOTUS domains found in Oskar, Tejas, and Tapas, but not MARF1, have a C-terminal extension, which is required for interaction with Vasa. Hence the LOTUS domains are divided into two subclasses: (1) extended LOTUS (eLOTUS) domain that is present in Oskar, Tejas, and Tapas, has a C-terminal extension, and binds Vasa, and (2) minimal LOTUS (mLOTUS) domain that is present in MARF1, lacks a C-terminal extension, does not bind Vasa, and instead binds the CCR4-NOT deadenylase complex. Thus, although eLOTUS and mLOTUS domains share core sequence homology except for the C-terminal extension, they mediate distinct protein-protein interactions. Interestingly, eLOTUS proteins (Oskar, Tejas/TDRD5, Tapas/TDRD7) contain a single eLOTUS domain while mLOTUS proteins (MARF1) contain multiple tandem mLOTUS domains (Zhu, 2018).

This study also showed that MARF1 binds cyclin A mRNA. In MARF1null mutant late-stage oocytes, cyclin A mRNA poly-A tail is longer and Cyclin A protein level is increased, without change in the cyclin A mRNA level. The degradation rate of Cyclin A protein was not changed in MARF1null oocytes compared with control oocytes in vitro. These results indicate that in control late-stage oocytes, MARF1 post-transcriptionally regulates Cyclin A protein level by binding cyclin A mRNA, shortening cyclin A mRNA poly-A tail, and reducing Cyclin A protein level. In contrast, poly-A shortening of cyclin A mRNA is lost in MARF1null oocytes, resulting in an accumulation of Cyclin A protein. Cyclin A is the only protein found that is increased in its level in MARF1 mutant oocytes (Zhu, 2018).

Based on these findings, a model is proposed for MARF1 molecular function. The MARF1 RRM binds specific target mRNAs, such as cyclin A mRNA, and the MARF1 mLOTUS domains recruit the CCR4-NOT deadenylase complex (see Models for MARF1 function in oocytes). This results in shortening of target mRNA poly-A tail and reduction of protein level produced from target mRNAs. The multiple, tandem mLOTUS domain may recruit multiple CCR4-NOT deadenylase complexes per single MARF1 molecule and single target mRNA, enabling efficient poly-A shortening (Zhu, 2018).

Using this model, it is proposed that MARF1 reduces Cyclin A protein level in stages 12-14 oocytes. This regulated reduction of Cyclin A protein level leads to expression of Cyclin B and Cyclin B3 proteins. This cyclin proteins expresson profile leads to stabilization of CDK1 protein and phosphorylation of CDK1 Tyr15 residue. Increase CDK1 and phosphorylation of CDK1 Tyr15 residue result in phosphorylation of appropriate target proteins of CDK1 in stage 14 oocytes. Proper global protein phosphorylation profile allows germplasm localization of Vasa and Aub and normal yolk distribution in stage 14 oocytes. As stage 14 oocytes traverse through the oviduct, meiotic Metaphase I arrest is released to complete meiosis and produce normal eggs (Zhu, 2018).

Consequently, it is speculated that Cyclin A is the main and/or most upstream target of MARF1. Persisted Cyclin A protein level in MARF1 mutant late-stage oocytes arrest them in an abnormal state rather than proceeding to a normal stage 14 including decreased protein levels of Cyclin B and Cyclin B3. Dysregulation of the three cyclin proteins levels results in the decreased CDK1 protein level and the decreased Tyr15 phosphorylation of CDK1. Dysregulation of cyclins and CDK1 alters global phosphorylation pattern. The altered phosphorylation pattern results in the loss of germplasm localization of Vasa and Aub. These together cause meiotic failure and complete sterility in MARF1 mutants (Zhu, 2018).

MARF1 seems to target specific mRNAs for gene silencing in diverse species. This study identified cyclin A mRNA as a target of Drosophila MARF1. Mouse MARF1 reduces protein and mRNA levels of retrotransposons and a few endogenous genes such as PPP2CB, suggesting that they are targets of the post-transcriptional silencing by mouse MARF1. Knockdown of human MARF1 causes upregulation of IFl44L mRNA, suggesting that IFl44L mRNA a target of human MARF1. Post-transcriptional gene silencing of target mRNAs in fly, mouse, and human MARF1 suggests that mLOTUS-domain directed recruitment of the CCR4-NOT deadenylase complex may be a widely conserved mechanism (Zhu, 2018).

LOTUS domains are found not only in animals but also in bacteria, fungi, and plants. LOTUS domains found in bacteria, fungi, and plants are more similar to the animal mLOTUS domains since they lack the C-terminal extension found in the animal eLOTUS domains. This suggests that the mLOTUS domain may be more ancient than the eLOTUS domain. It will be interesting to investigate the functions of these LOTUS domains found in non-animals, particularly the function of bacterial LOTUS domains, since bacteria do not have a poly-A tail in their mRNAs or CCR4-NOT deadenylase complex (Zhu, 2018).

The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis

CCR4-NOT is a highly conserved protein complex that regulates gene expression at multiple levels. In yeast, CCR4-NOT functions in transcriptional initiation, heterochromatin formation, mRNA deadenylation and other processes. The range of functions for Drosophila CCR4-NOT is less clear, except for a well-established role as a deadenylase for maternal mRNAs during early embryogenesis. This study report here that CCR4-NOT has an essential function in the Drosophila prothoracic gland (PG), a tissue that predominantly produces the steroid hormone ecdysone. Interfering with the expression of the CCR4-NOT components twin, Pop2, Not1, and Not3 in a PG-specific manner resulted in larval arrest and a failure to initiate metamorphosis. Transcriptome analysis of PG-specific Pop2-RNAi samples revealed that Pop2 is required for the normal expression of ecdysone biosynthetic gene spookier (spok) as well as cholesterol homeostasis genes of the NPC2 family. Interestingly, dietary supplementation with ecdysone and its various sterol precursors showed that 7-dehydrocholesterol and cholesterol completely rescued the larval arrest phenotype, allowing Pop2-RNAi animals to reach pupal stage, and, to a low degree, even survival to adulthood, while the biologically active hormone, 20-Hydroxyecdysone (20E), was significantly less effective. Also, genetic evidence is presented that CCR4-NOT has a nuclear function where CCR4-NOT-depleted cells exhibit aberrant chromatin and nucleoli structures. In summary, these findings indicate that the Drosophila CCR4-NOT complex has essential roles in the PG, where it is required for Drosophila steroid hormone production and cholesterol homeostasis, and likely has functions beyond a mere mRNA deadenylase in Drosophila (Zeng, 2018).

The CCR4-NOT complex is an evolutionarily conserved protein complex found in eukaryotes. The functions of CCR4-NOT have been linked to the regulation of gene expression at nearly all levels, including histone modifications, transcriptional initiation, transcriptional elongation, heterochromatin formation, translational repression as well as the regulation of mRNA degradation. Given the versatility of the CCR4-NOT complex, many aspects of its functions remain unclear. Most studies on CCR4-NOT have been conducted in Saccharomyces cerevisiae or human cell culture systems, with only a few studies that have examined this complex in multi-cellular organisms. In Drosophila, the main components of CCR4-NOT are conserved, which include the homologs of yeast carbon catabolite repression 4 (CCR4, encoded by twin), Pop2 (also known as CAF1), NOT1, NOT2 (encoded by Regena), NOT3 and CAF40 (encoded by Rcd1). Two of the components, CCR4/Twin and Pop2 hydrolyze mRNA poly(A) tails through their deadenylase activities, which leads to translational repression followed by mRNA decay. Two other Drosophila poly(A)-specific ribonucleases, the Pan2-Pan3 complex and the poly(A)-specific ribonuclease (PARN), have both specialized and redundant functions when compared to the CCR4-NOT complex, and together control overall mRNA stability. Most studies on Drosophila CCR4-NOT have only focused on its role in regulating mRNA stability, especially during oogenesis and early embryonic development before the midblastula transition. Before the midblastula transition, embryonic development relies exclusively on maternal mRNAs; hence there is almost no contribution from transcriptional regulation. Therefore, studying CCR4-NOT in the context of oogenesis and early embryogenesis provides only limited evidence as to whether the Drosophila CCR4-NOT complex has additional roles, such as transcriptional initiation or transcriptional elongation. While the CCR4-NOT complex can target a wide range of transcripts, a plethora of adapter proteins ensures context-specific degradation of select mRNA targets, allowing for tissue- and pathway-specific control. Genome-wide RNA interference (RNAi) screens conducted in two different biological contexts showed that CCR4-NOT complex function is essential for Drosophila neuroblast development where loss of Pop2 or NOT1 in neuroblasts resulted in animal lethality prior to the adult stage, while regulation of intestinal stem cell homeostasis is not dependent on CCR4-NOT function. Thus, CCR4-NOT function is highly regulated and differs drastically depending on biological context, and as such does not act as a global mRNA deadenylase. This study was interested in the specific roles of CCR4-NOT in the PG, which is a central endocrine tissue that coordinates multiple development events via the secretion of the steroid hormone ecdysone (Zeng, 2018).

Like all holometabolous insects, the Drosophila life cycle has discrete developmental stages, and pulses of ecdysone trigger all developmental transitions such as molting and metamorphosis. In Drosophila larvae, ecdysone is synthesized in the principal steroidogenic tissue, the PG, which is part of the ring gland, an endocrine organ composed of three glands. In the PG, tissue-specific expression of ecdysone biosynthetic enzyme genes including neverland (nvd), spookier (spok), shroud (sro), Cyp6t3, phantom (phm), disembodied (dib) and shadow (sad) allow the conversion of suitable sterols (e.g., cholesterol) to ecdysone. spok, sro, Cyp6t3, phm, dib and sad are commonly referred to as the Halloween genes. To ensure that these developmental transitions occur at the appropriate time, the PG acts as a decision-making center to integrate systemic and environmental cues into an ecdysone pulse that advances development when the proper conditions are met. This study reports a requirement for the CCR4-NOT complex for the production of ecdysone and maintaining cholesterol homeostasis. PG-specific RNAi against five out of seven CCR4-NOT components resulted in larval arrest mainly in the third instar (L3), a phenotype consistent with a block in ecdysone production. Transcriptional profiling via RNA-Seq of PG-specific Pop2-RNAi ring gland samples revealed that ecdysone and cholesterol pathways were particularly affected, and that cholesterol feeding could rescue the larval arrest phenotype of Pop2-RNAi animals, strongly supporting the idea that CCR4-NOT has specialized tissue-specific functions (Zeng, 2018).

To determine the importance of the CCR4-NOT complex in the PG, a PG-specific Gal4 driver (phm22-Gal4, referred to as PG>) to express UAS-RNAi transgenes targeting all seven CCR4-NOT subunits individually. For each gene, all available TRiP-RNAi lines from Bloomington Stock Center were tested, and the results from a previously published genome-wide PG-specific RNAi screen using VDRC RNAi lines was also included. For Pop2, two non-overlapping RNAi lines (PG>Pop2-RNAi1 and PG>Pop2-RNAi2) caused comparable developmental arrest phenotypes, which validated the specificity of the RNAi constructs. 100% of the PG>Pop2-RNAi1 and ~93% of the PG>Pop2-RNAi2 animals failed to pupariate, and remained 3rd instar (L3) larvae for several weeks, which resulted in substantially increased body sizes. This L3 arrest phenotype is commonly observed when the major ecdysone pulse that triggers the onset of metamorphosis fails to occur. Of the ~7% PG>Pop2-RNAi2 larvae that attempted puparium formation, none completed metamorphosis and thus failed to eclose as adults. As for the other CCR4-NOT components, at least one RNAi line targeting Twin, Not1, Not3, or Not4 was also found to cause L3 arrest. Taken together, these results indicated that the CCR4-NOT complex is essential for PG function, possibly by contributing to regulating ecdysone production (Zeng, 2018).

Given the potential requirement of the CCR4-NOT complex at nearly all levels of gene expression, transcriptional profiling of hand-dissected ring glands was performed via RNA-Sequencing (RNA-Seq). Ring glands from PG>Pop2-RNAi1 and PG>w1118 control animals were collected at 24 h after the 2nd to 3rd instar molt. This time point was chosed because ring glands from these larvae are large enough for dissection while larvae undergo minimal physiological changes compared to later time points closer to puparium formation. Pop2-RNAi1 was used to disrupt CCR4-NOT function because the RNAi phenotype was 100% penetrant and because the phenotypes were validated by a second independent RNAi line. For each sample, 50 ring glands, which should average out any individual timing differences between animals, were collected. Total RNA samples were enriched for mRNAs by selectively depleting rRNAs, followed by Illumina sequencing (Zeng, 2018).

The L3 arrest phenotype in Pop2-RNAi animals suggested a defect in ecdysone biosynthesis, and it was of interest to see whether the RNA-Seq results would reveal any reduction in the expression of the ecdysone biosynthetic enzyme genes. Interestingly, two genes critical for ecdysone biosynthesis were among the top 50 downregulated genes, namely spookier (spok) and neverland (nvd), ranked #31 (8.7-fold down) and #41 (7.7-fold down), respectively. However, only spok was confirmed to be significant (p < 0.05) for both the original RNA-Seq result and the qPCR validation data. For validation via qPCR, the weaker Pop2-RNAi2 line was used at an earlier developmental time point (0 h L3). Next, whether these changes in mRNA levels translated into corresponding reductions of protein levels of Nvd and Spok was investigated. It was found via immunostaining that Spok is strongly reduced in Pop2-RNAi1 PG cells, consistent with the mRNA expression levels. However, no significant reduction of Nvd was observed upon Pop2 knockdown. A possible explanation is that the net effect of a (albeit non-significant) reduction in nvd transcript levels vs. increased nvd transcript stability (due to loss-of-Pop2) results in overall similar protein levels. Alternatively, while the immunodetection and RNA-Seq experiments were conducted at the same developmental time point (96 h AEL = 24 h post L2/L3 molt), there may be a lag between reduction in transcript and protein levels due to protein perdurance. Significant transcriptional upregulation was also found for sro and dib via qPCR, while phm was not significantly changed. To test whether this corresponded to elevated protein levels in the PG, Sro and Phm were examined via immunofluorescence. Of the two, only Sro appeared to have moderately increased protein levels, again mirroring the mRNA expression data. It is concluded that the ecdysone deficiency phenotype caused by PG-specific RNAi of Pop2 was likely caused by a reduction in spok functiom (Zeng, 2018).

If Pop2 only functions as a mRNA deadenylase, one would expect a stabilization of direct target mRNAs in cells with impaired Pop2 function. Therefore, genes that register as downregulated in the RNA-Seq data, such as nvd and spok, are likely the result of indirect effects, or a combination of direct and indirect effects. Likewise, upregulated genes could be the result of direct stabilization via loss-of-Pop2 or again caused by direct and/or indirect effects. To distinguish these possibilities, poly(A) tail-length assays were carried out to test whether Pop2 depletion affects poly (A) tail lengths of six Halloween genes. For this, ring gland mRNAs were tagged with oligo-G/I bases followed by reverse-transcription into cDNA using the tag as the priming site. PCR was then performed by using a universal reverse primer complementary to the tag and a gene-specific primer, resulting in ~150-300 bp fragments. As controls, the same gene-specific primer was used coupled to a second downstream gene-specific primer that binds upstream of the poly (A) tail. This will give rise to an amplicon without the poly (A) tail, and allows estimation of the poly (A) tail length. Lengthening of the poly (A) tail was observed for spok, phm and dib transcripts in a Pop2-RNAi1 background relative to controls, indicating that these three Halloween transcripts are direct targets of Pop2. Since spok is showing a poly(A) upshift, but strongly downregulated both at the transcriptional and protein levels, it is concluded that while spok is a direct target of Pop2, there might a superimposed transcriptional effect in a Pop2 loss-of-function background. The results for nvd (repeated in two independents assays) were inconclusive. While nvd showed a clear upshift, a second band with little or no increased length was also present. In contrast, both sro and sad showed no lengthening of the poly(A) tail, indicating that the CCR4-NOT complex regulates only select Halloween transcripts (Zeng, 2018).

Since spok transcript levels are downregulated, two possibilities are seen that may explain this. First, it is conceivable that the CCR4-NOT complex acts directly as a transcriptional modulator of spok expression, in line with reported nuclear functions of CCR4-NOT in yeast. However, it is equally possible that mRNAs encoding transcriptional regulators of the Halloween genes are regulated by CCR4-NOT, and the resulting stabilization of such transcriptional regulators upon Pop2 knockdown would then indirectly alter Halloween gene expression. In fact, many such transcription factors have been identified, the most recent example being Kr-h1, which negatively regulates the transcription of the Halloween genes. Interestingly both nvd and spok are strongly downregulated by Kr-h1 overexpression, suggesting that both genes are coordinately regulated by this and possibly other repressors. To conclude, regardless of the complex regulation and possible crosstalk between the CCR4-NOT and transcriptional regulators, the net outcome of PG>Pop2-RNAi is the specific reduction of Spok (Zeng, 2018).

Nvd catalyzes the first step of ecdysone biosynthesis where it converts cholesterol to 7-dehydrocholesterol (7DC). Spok plays a crucial role in the 'Black Box', which consists of several not yet characterized conversion steps. However, the net result of the Black Box is that 5β-ketodiol (5βkd) is derived from 7DC. To test the idea that the ecdysone deficiency phenotype observed in PG>Pop2-RNAi animals was caused directly by reduced levels of spok and possibly nvd, fly media supplemented with either 7DC, 5βkd or the biologically active form of ecdysone (20E = 20-Hydroxyecdysone) was used. Remarkably, 7DC alone rescued the L3 arrest phenotype and enabled nearly 80% of larvae to reach the pupal stage. The rescue of the L3 arrest phenotype by 7DC was observed in both PG>Pop2-RNAi lines, demonstrating the specificity of the results. Intriguingly, compared to 7DC, the rescue by 20E (the end product of ecdysone biosynthesis) was much less pronounced and not significant in PG>Pop2-RNAi1 lines. Similarly, 20E did not rescue PG>Pop2-RNAi2 animals. Moreover, combining 7DC and 20E significantly lowered pupal survival compared to 7DC alone, suggesting that 20E has some adverse effect or was neutralizing 7DC efficiency in a PG>Pop2-RNAi background. It should be noted that 20E-supplementation rescues other Halloween gene loss-of-function animals (including PG>spok-, sro-, Cyp6t3-, phm-, dib- and sad-RNAi lines) very effectively and is not considered toxic per se. For instance, 20E can rescue the L2 arrest phenotype of PG>sro-RNAi larvae all the way to adulthood (~31% adult survival). Interestingly, it was recently reported that séance mutants selectively affect nvd expression, and can be effectively rescued with 7DC, but not 20E, consistent with the observation for Pop2-RNAi lines. When 5βkd-supplementation was used, no further improvement was seen, as most of the PG>Pop2-RNAi1 animals still could not develop beyond the larval stages. Thus, similar to 20E, rescue of PG>Pop2-RNAi animals via 5βkd was inferior to 7DC. A likely explanation for this is that 7DC feeds into pathways other than ecdysone production, and that Pop2 function is required for these undefined roles (Zeng, 2018).

Given that 7DC is a cholesterol metabolite, it is of interest to note that three Niemann-Pick disease type C (NPC) genes, npc2f, npc2d, and npc2c were upregulated 6.8-fold (ranked #41), 5.1-fold and 3.9-fold respectively in Pop2-RNAi samples compared to control ring gland samples. NPC proteins play critical roles in the intracellular transport of cholesterol and cholesterol metabolites, raising the idea that 7DC-supplementation compensates for this aberrant upregulation of NPC genes. Alternatively, NPC2 upregulation may compensate for an issue with cholesterol uptake or transport in Pop2-RNAi cells, an issue that could also be rescued by 7DC feeding since 7DC is the immediate metabolites of cholesterol. Consistent with the coordinated upregulation of three NPC2 genes, 'intracellular cholesterol transport' was identified as one of the functional association subgroups in the functional interaction analysis, where a subset of genes was used from RNA-Seq data with > 3-fold upregulation in Pop2-RNAi ring glands compared to controls. To test whether cholesterol supplementation was as effective as 7DC, feeding experiments were carried out using fly media supplemented with cholesterol. Remarkably, cholesterol rescued ~33% of the Pop2-RNAi1 animals to the pupal stage, and ~3% of the RNAi larvae were even rescued to adulthood, a result that could be confirmed with the second Pop2-RNAi line. Overall, cholesterol was as effective as 7DC in rescuing the larval arrest phenotypes, indicating that nvd is functioning normally in PG>Pop2-RNAi animals since Nvd is required for converting cholesterol to 7DC. In conclusion, three NPC2 genes linked to intracellular sterol transport appear to be misregulated upon Pop2-RNAi, indicating deregulation of cholesterol homeostasis, and consistent with this, both cholesterol and its immediate downstream metabolite, 7DC, are significantly more effective than 20E in rescuing PG-specific Pop2-depleted animals (Zeng, 2018).

When significantly altered genes were analyzed in the RNA-Seq datasets, it was noticed that the number of upregulated genes in PG>Pop2-RNAi ring glands were consistently higher than the downregulated genes, regardless of which cut-offs were used. For instance, the number of > 2.5-fold upregulated genes more than doubled the number of > 2.5-fold downregulated genes. This trend is consistent with the idea that Pop2 might primarily act to negatively regulate mRNA levels through deadenylation, and a decrease in RNA degradation would thus register as a higher percentage of upregulated genes (Zeng, 2018).

Next, functional association networks were generated of differentially expressed genes using STRING database to identify processes that are affected by the loss of Pop2 function. Genes were grouped within the interaction networks according to their gene ontology (GO) terms from DAVID GO and Flybase. For the 197 > 3.5-fold upregulated genes in the Pop2-RNAi ring gland compared to control samples, this analysis revealed genes associated with several functional groups: including 'transcription' (as well as 'splicing' and 'ribosome biogenesis'), 'mitochondrial translation', 'intracellular cholesterol transport', 'proteolysis', 'lipid catabolic process' and 'glycine/serine/threonine metabolism'. To complement this functional interaction analysis, gene ontology (GO) term and KEGG pathway enrichment analysis were performed via both DAVID GO and STRING database using a less stringent cut-off of > 2.5-fold upregulated genes (total 536 genes). Consistent with the results of the functional association study, genes that are involved in transcription as well as genes encoding mitochondrialribosomal proteins and mitochondrial respiratory chain complex III component were significantly overrepresented (Zeng, 2018).

For the total of 196 > 3-fold downregulated genes, several functional groups were also identified, including: 'translation', 'mitochondria', 'splice site/transcriptional start site selection', 'actin filament-related' and 'proteolysis'. The term enrichment analysis using the> 2-fold (total 296 genes) downregulated genes confirmed that genes involved in 'cytoplasmic translation' (5 out of 8 genes are ribosomal genes) and 'mitochondrial protein complex' were significantly enriched among differentially downregulated genes. Interestingly, two genes linked to 'cytoplasmic translation' encode proteins acting in the target of rapamycin (TOR) signaling pathway, namely Rheb and the eukaryotic translationinitiation factor (eIF) subunit 4E-binding protein (4E-BP) gene (Thor in Drosophila), which are both ~3.9-fold downregulated in Pop2-RNAi ring glands. This is intriguing because recently it was shown that TOR not only acts in amino acid sensing but is also activated by cellular cholesterol, consistent with the idea that CCR4-NOT has hitherto unknown links to cellular cholesterol homeostasis. In line with the misregulation in 4E-BP and Rheb, Pop2-RNAi ring glands displayed growth defects in a nutrient-dependent manner. Pop2-RNAi ring glands were normal when larvae were reared on yeast but had a reduced size when the Nutri-Fly Bloomington formula or a standard cornmeal-based recipe were used. It is pointed out that larvae with smaller ring glands had equal (standard cornmeal) or better survival rates (Nutri-Fly) compared to those with normal-sized ring glands (yeast) (Zeng, 2018).

When PG nuclei were analyzed via DAPI staining to assess the ring gland morphology, it was found that Pop2-RNAi animals exhibited uneven and fragmented DAPI signals, resulting in a region with reduced DAPI staining. In some nuclei, the chromatin was arranged in a way that resulted in a petal-like appearance. In wild-type cells, nuclear zones that stain weakly for DAPI correspond to the nucleolus, where rRNA synthesis and ribosomebiogenesis occurs. Since this observation was consistent with the functional association analysis ('ribosome biogenesis'), propidium iodide (PI) staining was carried out to visualize the nucleolus (because PI also binds RNA in addition to DNA). Interestingly, PI staining of Pop2-RNAi PG cells revealed some nucleoli that had a distinct 'hollow sphere' appearance, a phenotype that was never observed in controls. Chromatin fragmentation and compaction were also apparent in PI staining of Pop2-RNAi nuclei, consistent with the DAPI results. These nuclear phenotypes of Pop2-RNAi PG cells are consistent with finding in fission yeast that showed involvement of CCR4-NOT in heterochromatin formation. It, therefore, appears likely that Pop2 has a direct nuclear function that is critical for accessibility of the transcriptional machinery to DNA. It is concludeed that it is very likely that the Drosophila CCR4-NOT complex also has additional functions beyond its well-documented deadenylase activity (Zeng, 2018).

This study revealed that the function of the CCR4-NOT deadenylase complex is essential for ecdysone biosynthesis and a normal nuclear structure in the PG. Disrupting CCR4-NOT function specifically in the PG via Pop2-RNAi strongly reduced mRNA and protein levels of the ecdysone enzyme gene spok. Transcriptome analysis of a Drosophila tissue with impaired CCR4-NOT function is presented in this study. Term enrichment analysis of these genomic data suggested a defect in cholesterol homeostasis, which is consistent with the finding that the L3 arrest phenotype in PG>Pop2-RNAi animals was rescued by cholesterol and 7DC feeding. Moreover, Pop2-depletion in the PG appeared to affect the structure of both chromatin and the nucleolus, suggesting CCR4-NOT also has nuclear functions, consistent with previous reports in yeast. It is concluded that CCR4-NOT has tissue-specific roles and is likely a multifunctional protein complex that has functions beyond the mRNA deadenylase in Drosophila (Zeng, 2018).

twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis: Twin regulates Cyclin A translation

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oogenesis, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. This study reports the identification of twin, a homolog of the yeast CCR4 deadenylase. twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, these data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects observed in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. It is proposed that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation (Morris, 2005).

twin encodes the Drosophila homolog of the yeast ccr4 gene. ccr4 (carbon-catabolite-repression) was first identified in S. cerevisiae as a regulator of RNA levels of the alcohol-dehydrogenase II gene. Although CCR4 protein was previously shown to associate with basal transcription machinery, recent data demonstrate that CCR4 catalyzes the degradation of poly(A) tails in yeast and flies (Temme, 2004; Morris, 2005 and references therein).

It has been unclear whether mutations in CCR4 have specific developmental defects and whether these defects might reveal specific targets sensitive to CCR4 function. twin mutant cysts divide asynchronously and less than four times; oocyte specification is defective and many egg chambers die and degrade. The mitotic cyclins, CycA and CycB, are misexpressed in twin, and reducing the gene copy number of cycA partially suppresses the twin egg chamber degradation phenotype. Furthermore, the poly(A) tails of cycA, cycE and, to a lesser extent, cycB, are longer in twin extracts, suggesting that Twin/Ccr4 deadenylase activity directly controls the RNA levels of these cell cycle regulators. By contrast, cytoplasmic Bam staining is reduced in twin. Induction of extra bam expression suppresses the cyst division and oocyte fate specification defects in twin mutants, implicating low Bam levels as one of the causes of these twin phenotypes (Morris, 2005).

The twin alleles are viable and specifically affect the female germline. In S. cerevesiae, ccr4 mutations are not lethal, although CCR4 is thought to be the main cytoplasmic deadenylase. It is possible that angel and Dnocturnin (CG4796), two other genes with extensive homology to the ccr4 catalytic domain but lacking the crucial LRR repeats, can partially compensate for loss of Twin function. Alternatively, since the mutations are probably not complete nulls, oogenesis may be more sensitive than the soma to decreased Twin function. Like the ovary, the early embryo relies on precise post-transcriptional gene regulation. The mature egg contains high levels of maternally loaded twin, consistent with a role for Twin in deadenylation, and probably explaining why twin mutants carry out embryogenesis normally (Morris, 2005).

Mitotic cells regulate cyclin levels in order to progress through the cell cycle. At the protein level, Drosophila regulates CycA, CycB and CycE, via proteasome-mediated degradation. In the Drosophila ovary, the novel protein Encore has been proposed to localize components of the proteasome complex to the fusome to regulate CycE. encore mutant cysts undergo an extra cell division and contain 32 cells, probably as a consequence of misexpressing not only CycE, but also CycA. Other experiments have shown that cyst divisions are sensitive to CycA levels. Adding a brief pulse of CycA by inducing a heat-shock construct can lead to an extra round of cyst division, suggesting that downregulation of CycA is crucial for cell cycle progression. Only a small number of cysts respond to such a CycA pulse, suggesting that in the wild type not all germ cells are in a susceptible phase of the cell cycle (G2) during which they can respond to CycA (Morris, 2005).

Cyclin RNA levels are regulated by control of poly(A) tail length. In Xenopus and mouse oocytes, cycB RNA is not translated in the absence of CPEB-mediated poly(A) tail lengthening. Longer poly(A) tails also enhance cyclin translation in Drosophila embryos. In the Drosophila ovary, Orb, the CPEB homolog, regulates poly(A) tail length and expression of its own RNA and oskar RNA. Consistent with a role for Orb in cyclin regulation and cyst division, orb mutant cysts frequently contain eight germ cells (Morris, 2005).

The data suggest that Twin-mediated deadenylation of cyclin RNA regulates cyst divisions. Cyclin polyadenylation has been well studied, but much less is known about cyclin RNA deadenylation. In Drosophila, Nanos and Pumilio have been shown to control deadenylation of cycB mRNA in primordial germ cells. Furthermore, Xenopus Pumilio interacts with CPEB, and Nanos, Pumilio and Orb/CPEB are all expressed early in Drosophila oogenesis. It is intriguing to speculate that Twin may regulate the poly(A) tail lengths in the dividing cyst in conjunction with Nanos, Pumilio and/or Orb (Morris, 2005).

Cytoplasmic Bam expression is reduced in twin germaria; a phenotype that would not be predicted if Twin directly regulated Bam expression via deadenylation. Indeed, no substantial change was detected in bam poly(A) tail length in twin ovaries. It is therefore proposed that bam is an indirect target of Twin/Ccr4 (Morris, 2005).

Although bam is known to control the differentiation of the cystoblast and to promote cyst division, the biochemical role of Bam is unknown. Removing one copy of bam suppresses the extra division in cysts lacking encore or overexpressing CycA. The results further implicate Bam in the events of early oogenesis. Increased bam expression suppresses not only the cyst division defects observed in twin mutants, but also the twin oocyte specification defects. Because Twin regulates cycA directly and may regulate Bam indirectly, the simplest model would posit that high levels of CycA are sufficient to suppress Bam expression. Two pieces of evidence argue against this model: Bam and CycA are both present at high levels in the dividing cyst; and Bam is required for the fifth cyst division induced by high levels of CycA. In addition, hs-bam induces stem cells to develop into normal cysts, indicating that high Bam levels do not disrupt CycA expression. A model is favored by which Bam and CycA act in parallel to each other, downstream of Twin (Morris, 2005).

Although several models could explain the data, it is proposed that increased mitotic cyclin levels together with low Bam expression cause many of the twin phenotypes. If Bam expression were normal, overexpressing cyclins could lead to extra cyst divisions. The low level of Bam in twin germaria does not permit continued cell division, yet cyclin levels remain high, delaying cell cycle progression and probably causing the egg chamber degradation observed in twin. This model is consistent with the fact that reducing the copy number of bam suppresses the extra cyst division phenotype of encore and of hs-cycA. Corroborating evidence comes from the observation that reducing the gene dose of cycA or increasing the dose of bam can partially suppress the degradation phenotype. However, there are likely to be other, unidentified targets of twin that also contribute to the twin phenotype (Morris, 2005).

twin and Hu Li Tai Shao mutants disrupt the number and synchrony of cyst divisions and oocyte specification. This array of defects is not shared by the cell cycle mutants described above or by other mutants such as orb, the M-phase inhibitor tribbles or the M-phase activator string, which affect the number but not the synchrony of cyst divisions. Comparison of twin and hts may therefore be instructive. hts cysts have no fusome, and are thought consequently not to coordinate the cyst divisions. By contrast, cysts in twin mutants contain branched fusomes that are capable of colocalizing with CycA, suggesting the possibility that Twin/Ccr4 gene regulation may mediate the coordination of the cyst divisions with oocyte specification downstream of the fusome (Morris, 2005).

mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the RNA-binding protein GW182, which interacts with the AGOs and is required for miRNA function. Depletion of Drosophila GW182 (Gawky), leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1 and NOT1, components of a deadenylase complex, or the DCP1:DCP2 decapping protein complex. The N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. These findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets (Behm-Ansmant, 2006).

To accomplish their regulatory function miRNAs associate with the Argonaute proteins to form RNA-induced silencing complexes (RISCs), which elicit decay or translational repression of complementary mRNA targets. In plants, miRNAs are often fully complementary to their targets, and elicit mRNA decay. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that remain elusive. Recent studies have shown that miRNAs silence gene expression by inhibiting translation initiation at an early stage involving the cap structure; mRNAs translated via cap-independent mechanisms escape miRNA-mediated silencing. Other studies have suggested that translation inhibition occurs after initiation, based on the observation that miRNAs and some targets remain associated with polysomes. In addition, animal miRNAs can also induce significant degradation of mRNA targets despite imperfect mRNA-miRNA base-pairing (Behm-Ansmant, 2006 and references therein).

The existence of a link between the miRNA pathway and mRNA decay is supported by the observation that mammalian Argonaute proteins (AGO1-AGO4), miRNAs, and miRNA targets colocalize to cytoplasmic foci known as P-bodies. These mRNA processing bodies are discrete cytoplasmic domains where proteins required for bulk mRNA degradation in the 5'-to-3' direction accumulate (e.g., the decapping DCP1:DCP2 complex and the 5'-to-3' exonuclease XRN1). Additional components of P-bodies in yeast and/or human cells include the CCR4:NOT deadenylase complex, auxiliary decapping factors (e.g., the LSm1-7 complex and Pat1p/Mtr1p), the cap-binding protein eIF4E, and the RNA helicase Dhh1/Me31B involved in translational repression. In metazoa, P-bodies are also marked by the presence of GW182, a protein with glycine-tryptophan repeats (GW repeats) required for P-body integrity (Behm-Ansmant, 2006 and references therein).

The presence of Argonaute proteins, miRNAs, and miRNA targets in P-bodies has led to a model in which translationally silenced mRNAs are sequestered to these bodies, where they may undergo decay. At present, it is unclear whether the localization in P-bodies is the cause or consequence of the translational repression, though several lines of evidence point to a direct role for P-body components in miRNA-mediated gene silencing. (1) DCP1, GW182, and its paralog TNRC6B associate with AGO1 and AGO2 in human cells; (2) depletion of GW182 in human cells impairs both miRNA function and mRNA decay triggered by complementary short interfering RNAs (siRNAs). Similarly, miRNA function is impaired in Drosophila Schneider cells (S2 cells) depleted of GW182 or the decapping DCP1:DCP2 complex (Rehwinkel, 2005). (3) The Caenorhabditis elegans protein AIN-1, which is related to GW182, is required for gene regulation by at least a subset of miRNAs (Behm-Ansmant, 2006 and references therein).

In Drosophila, siRNA-guided endonucleolytic cleavage of mRNAs (RNA interference [RNAi]) is mediated by AGO2, while gene silencing by miRNAs is mediated by AGO1. That siRNAs and miRNAs enter separate pathways in Drosophila is further supported by the observation that depletion of GW182 inhibits miRNA-mediated, but not siRNA-mediated gene silencing (Rehwinkel, 2005). The precise role of GW182 in the miRNA pathway is unknown. GW182 could have an indirect role by affecting P-body integrity. Alternatively, it could be more directly involved, localizing miRNA targets to P-bodies or facilitating the mRNP remodeling steps required for the silencing and/or decay of these targets (Behm-Ansmant, 2006 and references therein).

This study investigates the role of Drosophila GW182 in the miRNA pathway. Depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of AGO1, indicating that GW182 is a genuine component of the miRNA pathway. In cells in which miRNA-mediated gene silencing is suppressed by depletion of AGO1, GW182 can still silence the expression of bound mRNAs, suggesting that GW182 acts downstream of AGO1. It is further shown that GW182 triggers silencing of bound transcripts by inhibiting protein expression and promoting mRNA decay via a deadenylation and decapping mechanism. Finally, evidence is provided that mRNA degradation by miRNAs requires GW182, the CCR4:NOT deadenylase, and the DCP1:DCP2 decapping complexes. Together with the observation that GW182 interacts with AGO1, these results indicate that binding of GW182 to miRNA targets induces silencing and can trigger mRNA degradation, providing an explanation for the observed changes in mRNA levels, at least for a subset of animal miRNA targets (Behm-Ansmant, 2006).

These results indicate that GW182 is a genuine component of RNA silencing pathways, associating with the Argonaute proteins and with components of the mRNA decay machinery and, providing a molecular link between RNA silencing and mRNA degradation. Depletion of GW182 or AGO1 from Drosophila cells leads to correlated changes in mRNA expression profiles, indicating that these proteins act in the same pathway. Transcripts commonly up-regulated by AGO1 and GW182 are enriched in predicted and validated miRNA targets. These results, together with the observation that GW182 associates with AGO1, identify GW182 as a component of the miRNA pathway (Behm-Ansmant, 2006).

GW182 belongs to a protein family with GW repeats, a central UBA domain, and a C-terminal RRM. Multiple sequence alignment of all proteins possessing these domains revealed that there are three paralogs (TNRC6A/GW182, TNRC6B, and TNRC6C) in vertebrates, a single ortholog in insects, and no orthologs in worms or fungi. At present, it is unclear whether the vertebrate paralogs have redundant functions, but both GW182 and TNRC6B have been shown to associate with human AGO1 and AGO2 (Behm-Ansmant, 2006).

In Drosophila, GW182 interacts with AGO1 in vivo and in vitro. No stable interaction with AGO2 was detected under the same conditions, suggesting that AGO2 may act independently of GW182. This is consistent with the observation that depletion of GW182 does not affect siRNA-guided mRNA cleavage or RNAi, which is mediated exclusively by AGO2 in Drosophila. Nevertheless, since AGO2 also regulates the expression levels of a subset of miRNA targets (Rehwinkel, 2006), the lack of interaction with GW182 raises the question of whether this regulation occurs by a similar or different mechanism from that mediated by AGO1. Further studies are needed to elucidate the mechanism by which Drosophila AGO2 regulates the expression of a subset of miRNA targets (Behm-Ansmant, 2006).

The N-terminal GW repeat region of GW182 encompasses two highly conserved motifs (I and II) and is expanded in vertebrates. This region is shorter in insects and bears similarity to the GW-like regions in the C. elegans protein AIN-1, involved in the miRNA pathway. However, AIN-1 does not contain UBA, Q-rich, or RRM domains. This lack of common domain architecture suggests that AIN-1 represents a functional analog. Nevertheless, the observation that C. elegans AIN-1 also localizes to P-bodies and interacts with AGO1 (i.e., worm ALG-1), and the finding that the N-terminal GW repeats of Drosophila GW182 interact with the PIWI domain of AGO1, suggest a conserved role for these repeats in mediating the interaction with Argonaute proteins. It would be of interest to determine the molecular basis of the specific interaction between the N-terminal GW repeats of GW182 and the PIWI domain of AGOs, and whether this interaction affects the catalytical activity of the domain (Behm-Ansmant, 2006).

Apart from the interaction with AGO1, the N-terminal repeats and the UBA and Q-rich domains contribute to the localization of GW182 in P-bodies, which is in turn required for P-body integrity. This suggests that GW182 may act as a molecular scaffold bringing together AGO1-containing RISCs and mRNA decay enzymes, possibly nucleating the assembly of P-bodies. Understanding the precise role of the various GW182 domains in the interaction with mRNA decay enzymes and AGO1 as well as in P-body integrity awaits further biochemical characterization (Behm-Ansmant, 2006).

Tethering GW182 to a reporter transcript silences its expression, bypassing the requirement for AGO1. Silencing by GW182 occurs by two distinct mechanisms: repression of protein expression, and mRNA degradation. It remains to be elucidated how GW182 represses translation. mRNA degradation by GW182 is inhibited in cells depleted of CAF1, NOT1, or the DCP1:DCP2 complex, indicating that GW182 promotes mRNA deadenylation and decapping. Thus, binding of GW182 appears to be a point of no return, which marks transcripts as targets for degradation (Behm-Ansmant, 2006).

More studies are needed to determine whether decapping triggered by GW182 requires prior deadenylation or whether these two events occur independently. The observation that mRNA levels are fully restored in cells depleted of DCP1:DCP2, suggests that deadenylation followed by 3'-to-5' exonucleolytic degradation is unlikely to represent a major pathway by which these mRNAs are degraded. Future studies should also reveal the identity of the nuclease(s) acting downstream of the decapping enzymes (Behm-Ansmant, 2006).

Previous studies indicate that miRNAs can reduce the levels of the targeted transcripts, and not just the expression of the translated protein. Consistently, transcripts up-regulated in cells depleted of AGO1 or GW182 are enriched in predicted and validated miRNA targets. In this paper further evidence is provided indicating that miRNAs silence gene expression by two mechanisms: one mechanism involving translational silencing, and one involving mRNA degradation. The contribution of these mechanisms to miRNA-mediated gene silencing appears to differ for each miRNA:target pair. Indeed, of the three reporters analyzed, Nerfin is silenced mainly at the translational level, silencing of the CG10011 reporter can be attributed to mRNA degradation, while Vha68-1 is regulated both at the translational and mRNA levels. Regardless of the extent of the contribution of these two mechanisms to silencing, both require AGO1 and GW182, because the levels of the mRNA reporter and luciferase activity are restored in cells depleted of any of these two proteins (Behm-Ansmant, 2006).

In contrast, although the levels of the mRNA reporter are restored in cells depleted of CAF1 or NOT1, translational repression is not fully relieved, indicating that deadenylation is required for mRNA decay, but not for translational silencing by miRNAs. In agreement with this, two reports published while this manuscript was in preparation have shown that miRNAs trigger accelerated deadenylation of their targets (Giraldez, 2006; Wu, 2006). This study extends these observations further by demonstrating: (1) deadenylation is mediated by the CCR4:NOT complex; (2) decapping is also required for miRNA target degradation, and (3) both deadenylation and decapping triggered by miRNAs requires GW182 (Behm-Ansmant, 2006).

Based on the results presented in this study and the observations that GW182 associates with AGO1 and is required for miRNA-mediated gene silencing, the following model is proposed: AGO1-containing RISCs binds to mRNA targets by means of base-pairing interactions with miRNAs; AGO1 may then recruit GW182, which marks the transcripts as targets for decay via a deadenylation and decapping mechanism (Behm-Ansmant, 2006).

A question that remains open is whether miRNA-mediated translational repression is the cause of mRNA degradation or whether these represent two independent mechanism by which miRNAs silence gene expression as proposed by Wu (2006). Indeed, changes in mRNA levels are not observed for all miRNA targets (Rehwinkel, 2006), suggesting that inhibition of translation is not always followed by mRNA decay. Conversely, depletion of CAF1 or NOT1 prevents mRNA decay but does not relieve translational silencing, suggesting that these two processes are independent (Behm-Ansmant, 2006).

An important finding is that miRNAs elicit degradation to different extents. One possible explanation is that the extent of degradation depends on the stability of the miRNA:mRNA duplexes. Also, the extent of degradation might depend on the particular set of proteins associated with a given target. For instance, some targets may assemble with a set of proteins that antagonize degradation. Finally, GW182 might interact only with a subset of AGO1-containing RISCs, as suggested for AIN-1. A major challenge will be to identify the specific features of miRNA targets and/or RISC complexes that lead to regulation of gene expression at the level of translation or at the level of mRNA stability (Behm-Ansmant, 2006).

Smaug recruits the CCR4/POP2/NOT deadenylase complex to trigger maternal transcript localization in the early Drosophila embryo

Asymmetric localization of mRNAs within cells promotes precise spatio-temporal control of protein synthesis. Although cytoskeletal transport-based localization during Drosophila oogenesis is well characterized, little is known about the mechanisms that operate to localize maternal RNAs in the early embryo. One such mechanism -- termed 'degradation/protection' -- acts on maternal Hsp83 transcripts, removing them from the bulk cytoplasm while protecting them in the posterior pole plasm. The RNA binding protein, Smaug, previously known as a translational repressor of nanos, has been identified as a key regulator of degradation/protection-based transcript localization. In smaug mutants, degradation of Hsp83 transcripts is not triggered, and, thus, localization does not occur. Hsp83 transcripts are in an mRNP complex containing Smaug, but Smaug does not translationally repress Hsp83 mRNA. Rather, Smaug physically interacts with the CCR4/POP2/NOT deadenylase, recruiting it to Hsp83 mRNA to trigger transcript deadenylation and degradation. When Smaug is targeted to heterologous stable reporter transcripts in vivo, these are deadenylated and destabilized. A deletion that removes the gene encoding CCR4 exhibits dose-sensitive interactions with Smaug in both a loss-of-function and a gain-of-function context. Reduction of CCR4 protein levels compromises Hsp83 transcript destabilization. It is concluded that Smaug triggers destabilization and localization of specific maternal transcripts through recruitment of the CCR4/POP2/NOT deadenylase. In contrast, Smaug-mediated translational repression is accomplished via an indirect interaction between Smaug and eIF4E, a component of the basic translation machinery. Thus, Smaug is a multifunctional posttranscriptional regulator that employs distinct mechanisms to repress translation and to induce degradation of target transcripts (Semotok, 2005; full text of article).

Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4

Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. This study identifies a novel level of nanos translational control. The Smaug protein bound to the nanos 3' UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. A mechanism is proposed for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development (Zaessinger, 2006).

Post-transcriptional mechanisms of gene regulation play a prominent role during early development. Because the oocyte and developing embryo go through a phase in which no transcription takes place, gene expression relies on a pool of maternal mRNAs accumulated during oogenesis and is regulated at the level of translation or mRNA stability. It has been shown in several biological systems that poly(A) tail shortening contributes to translational silencing, whereas translational activation requires poly(A) tail extension. Poly(A) tail shortening, or deadenylation, is also the first step in mRNA decay. Subsequent steps occur only after the poly(A) tail has been shortened beyond a critical limit. Rapid deadenylation of unstable RNAs is caused by destabilizing elements, for example AU-rich elements (AREs) found in the 3' UTRs of several mRNAs. A number of proteins have been identified that bind to destabilizing RNA sequences and accelerate deadenylation as well as subsequent steps of decay (Zaessinger, 2006).

In yeast, deadenylation is mostly catalyzed by the multi-subunit CCR4-NOT complex, and this complex is also involved in deadenylation in Drosophila (Temme, 2004) and in mammalian cells. A second conserved deadenylase, the heterodimeric PAN2-PAN3 complex, appears to act before the CCR4-NOT complex. A third enzyme, the poly(A)-specific ribonuclease (PARN) is present in most eukaryotes but has not been found in yeast and Drosophila (Zaessinger, 2006).

Translational regulation of maternal mRNAs in Drosophila is essential to the formation of the anteroposterior body axis of the embryo. During embryogenesis, a gradient of the Nanos (Nos) protein arises from the posterior pole and organizes abdominal segmentation. This gradient results from translational regulation of maternal nos mRNA. The majority of nos transcripts is uniformly distributed throughout the bulk cytoplasm and is translationally repressed and subsequently degraded during the first 2-3 hours of embryonic development. A small proportion of nos transcripts is localized in the pole plasm, the cytoplasm at the posterior pole that contains the germline determinants. This RNA escapes repression and degradation, and its translation product forms a concentration gradient from the posterior pole. Both translation activation at the posterior pole and repression elsewhere in the embryo are essential for abdominal development, and head and thorax segmentation, respectively (Zaessinger, 2006 and references therein).

Translation of nos mRNA is repressed in the embryo by Smaug (Smg), which binds two Smaug response elements (SREs) in the proximal part of the nos 3' UTR. The SREs are also essential for the decay of nos mRNA. Repression of nos translation appears to be a multistep process, involving at least one level of regulation at the initiation step and another after nos mRNA has been engaged on polysomes. Repression at the initiation step is thought to involve an interaction between Smg and the protein Cup. The latter associates with the cap-binding initiation factor eIF4E, displacing the initiation factor eIF4G. Translation of nos mRNA at the posterior pole depends on Oskar (Osk) protein, although its mechanism of action has remained unknown (Zaessinger, 2006).

Bulk nos mRNA has a short poly(A) tail, and it was thought that nos translational control was independent of poly(A) tail length regulation. More recently, Smg and its yeast homologue Vts1 were shown to be involved in the degradation of mRNAs. Smg induces degradation and deadenylation of Hsp83 mRNA during early embryogenesis. This appears to result from recruitment by Smg of the CCR4-NOT deadenylation complex on Hsp83 mRNA, although the Smg-binding sites in this mRNA have not been identified. However, Hsp83 mRNA deadenylation was reported not to repress its translation. This study shows that nos mRNA is subject to regulation by active deadenylation by the CCR4-NOT deadenylation complex. This deadenylation depends on Smg and on the SREs in the 3' UTR of nos mRNA. The model is confirmed of the CCR4-NOT complex recruitment by Smg, in this case, onto nos mRNA, using genetic interactions between mutants affecting smg and the CCR4 deadenylase, and showing the presence in a same protein complex of endogenous Smg and CAF1, a protein of the CCR4-NOT complex. Active deadenylation of nos mRNA contributes to its translational repression in the bulk embryo and is essential for the anteroposterior patterning of the embryo. Moreover, Osk activates translation of nos by preventing the specific binding of Smg protein to nos mRNA, thereby precluding active deadenylation and destabilization of nos mRNA (Zaessinger, 2006).

This paper shows that poly(A) tail length regulation is central to nos translational control. Poly(A) tail length regulation is a major mechanism of translational control, particularly during early development. nos translational control has been reported to be independent of poly(A) tail length. This conclusion came from the absence of nos poly(A) tail elongation between ovaries and early embryos, and the lack of nos poly(A) tail shortening between wild-type and osk mutant embryos in which nos mRNA is not translated at the posterior pole. However, later studies suggested that this lack of poly(A) tail change was not unexpected, as nos mRNA translation starts in ovaries, and the pool of translationally active nos mRNA in embryos is very small (4%) and remains undetected among the amount of translationally repressed nos in whole embryos. It has now been found that nos mRNA deadenylation by the CCR4-NOT complex, recruited to the 3' UTR by Smg, is required for nos translational repression in the bulk embryo. In addition, these data also suggest that nos translation at the posterior pole depends on the prevention of this deadenylation. nos mRNA is regulated at several levels, including localization, degradation, translational repression and translational activation. Localization at the posterior pole depends on two mechanisms: an actin-dependent anchoring at late stages of oogenesis, after nurse cells dumping and localized stabilization. Localization and translational control are coupled in that the localized RNA escapes both translational repression and degradation. A mechanism is proposed for this coupling. Translational repression and RNA degradation both involve Smg-dependent deadenylation. Deletion of the SREs in a nos transgene, as well as mutations in smg or in twin, which encodes the major catalytic subunit of the deadenylating CCR4-NOT complex, abrogate poly(A) tail shortening. Lack of deadenylation prevents the timely degradation of the RNA and also relieves translational repression. Deadenylation could repress nos mRNA translation by two mechanisms. Interaction of the cytoplasmic poly(A) binding protein (PABP) with mRNA poly(A) tails is important for the activation of translation initiation. Therefore, poly(A) shortening of nos mRNA would lead to PABP dissociation and inhibition of translation. In addition, deadenylation leads eventually to nos mRNA decay, which should also contribute to translational repression. Consistent with the Smg-dependent deadenylation of nos mRNA, describe in embryos, a recent study documented SRE-dependent deadenylation of chimeric transcripts containing the 3' UTR of nos mRNA in cell-free extracts from Drosophila embryos (Jeske, 2006). In this system, deadenylation of the chimeric RNAs also strongly contributes to translational repression, along with at least another deadenylation-independent mechanism (Zaessinger, 2006).

In this analysis, twin and smg mutants, although both impaired in nos mRNA poly(A) tail shortening, did not show the same defects. twin mutants fail to show nos poly(A) tail shortening during embryogenesis, whereas in smg mutant embryos or when poly(A) tails are measured from nos(ΔTCE) transgene, a poly(A) tail elongation is visible. This suggests that nos mRNA is also regulated by cytoplasmic polyadenylation which balances the deadenylation reaction, and that Smg binding to the RNA reduces the polyadenylation reaction. Consistent with a dynamic regulation of poly(A) tail length of maternal mRNAs resulting from a tight balance between regulated deadenylation and polyadenylation, it was found that in mutants for the GLD2 poly(A) polymerase, which is involved in cytoplasmic polyadenylation, nos mRNAs are precociously degraded in 0-1 hour embryos (Zaessinger, 2006).

Ectopic expression of osk in the bulk cytoplasm of the embryo is sufficient to impair nos mRNA binding to Smg and its deadenylation and destabilization. Therefore, it is proposed that, in wild-type embryos, Osk at the posterior pole inhibits Smg binding to the anchored nos mRNA, preventing deadenylation, decay and translational repression. This results in localized nos stabilization and translation. Osk might achieve this by a direct binding to Smg; it was shown to interact with Smg in vitro, through a region overlapping the RNA-binding domain in Smg. Alternatively, Osk could prevent Smg function independently of its binding to Smg, through its recruitment by another protein in nos-containing mRNPs. Consistent with a potential presence of Smg and Osk in the same protein complex, it was possible to co-immunoprecipitate Osk with Smg in embryos overexpressing Osk (Zaessinger, 2006).

Two mechanisms of nos translational repression have already been described. A first mode of translation inhibition appears to act during elongation, as suggested by polysome analysis and by the involvement of the Bicaudal protein, which corresponds to a subunit of the nascent polypeptide associated complex. The second mode of repression involves Smg and is thought to affect initiation. It requires the association of Smg with the protein Cup, which also binds eIF4E. The association of Cup with eIF4E competes with the eIF4E/eIF4G interaction, which is essential for translation initiation. This study identifies deadenylation by the CCR4-NOT complex as a novel level of nos translational repression, also involving Smg. Smg protein synthesis is probably induced by egg activation during egg-laying. Smg is absent in ovaries and accumulates during the first hours of embryogenesis, with a peak at 1-3 hours. Its amount is low during the first hour and possibly nonexistent during the first 30 minutes. This correlates with the presence at that time of high levels of nos mRNA in the bulk embryo that are not destabilized. nos translational repression is active, however, as this pool of mRNA is untranslated. Thus a Smg-independent mode of repression must be efficient during the first hour of development. This might correspond to repression at the elongation step and/or involve the Glorund protein, a Drosophila hnRNP F/H homologue newly identified as a nos translational repressor in the oocyte (Kalifa, 2006). Glorund has a role in repression of unlocalized nos mRNA in late oocytes and has been suggested to also act at the beginning of embryogenesis while Smg is accumulating to ensure the maintenance of translational repression at the oogenesis to embryogenesis transition (Kalifa, 2006). Analysis of glorund mutants revealed that the embryonic phenotypes are less severe than expected and led to the proposal that at least an additional level of nos translational repression is active in oocytes (Kalifa, 2006). Overexpression of Osk in the germline with nos-Gal4 results in long poly(A) tails of nos mRNA, even in 0-1 hour embryos in which Smg protein is poorly expressed. This suggests that the short poly(A) tail of nos mRNA in 0-1 hour wild-type embryos could in part result from active deadenylation during oogenesis, which would depend on a regulatory protein different from Smg. Deadenylation could therefore be involved in nos regulation during oogenesis, and would also be prevented by Osk in the pole plasm, as in embryos (Zaessinger, 2006).

Genetic evidences indicate that all three levels of translational repression are additive. Although the importance of the Smg/Cup/eIF4E mode of nos translational repression for the anteroposterior patterning of the embryo has not been addressed, the other two levels of repression are essential, as ectopic Nos protein leads to disruption of the embryo anteroposterior axis in twin or bicaudal mutants. This demonstrates that none of the three levels of repression is sufficient by itself and suggests that all three regulations are required to achieve complete translational repression of nos. As Osk acts by preventing the binding of Smg to the nos 3' UTR, it is likely to inhibit both Smg-dependent mechanisms of translational repression (Zaessinger, 2006).

The presence of Smg in discrete cytoplasmic foci and its partial colocalization in these foci with components of the CCR4-NOT deadenylation complex, and with components of P bodies, suggest that Smg-dependent deadenylation and translational control of nos occur in P bodies. P body dynamics and function have not been addressed in a complete organism during development. Consistent with the apparent complexity of P body function, including mRNA decay and translational repression, embryos different subsets of Smg-containing structures were identified in embryos: these subsets either do or do not contain the CCR4-NOT deadenylation complex and the Xrn1 5'-3' exonuclease. This suggests the existence of different types of P bodies that may have distinct functions (Zaessinger, 2006).

The CCR4-NOT complex is involved in default deadenylation of bulk mRNAs in somatic cells (Temme, 2004). This study finds that the same deadenylation complex has a role in active, sequence-specific deadenylation of a particular mRNA. Activation of deadenylation by CCR4-NOT results from the recruitment of the deadenylation complex by a regulatory RNA-binding protein to its specific mRNA target (this study; Semotok, 2005). Several RNA-binding proteins are expected to interact with the CCR4-NOT complex to regulate the deadenylation of different pools of mRNAs in different tissues. CCR4 controls poly(A) tail lengths of Cyclin A and B mRNAs during oogenesis (Morris, 2005); the regulatory protein has not been identified, but it cannot be Smg, which is not expressed in ovaries. A similar mode of active deadenylation involving the recruitment of the deadenylation complex by ARE-binding proteins has been proposed in mammalian cells. A study in yeast has identified the PUF (Pumilio/FBF) family of RNA-binding proteins as activators of CCR4-NOT-mediated deadenylation through a direct interaction between PUF and POP2 (the CAF1 homologue). Although default deadenylation by CCR4 is not essential for viability (Temme, 2004), active deadenylation by CCR4 of specific mRNAs is essential for certain developmental processes, in particular during early development (Zaessinger, 2006).

Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression

Bicaudal C (Bic-C) encodes an RNA-binding protein required maternally for patterning the Drosophila embryo. A set of mRNAs have been identified that associate with Bic-C in ovarian ribonucleoprotein complexes. These mRNAs are enriched for mRNAs that function in oogenesis and in cytoskeletal regulation, and include Bic-C RNA itself. Bic-C binds specific segments of the Bic-C 5' untranslated region and negatively regulates its own expression by binding directly to NOT3/5, a component of the CCR4 core deadenylase complex, thereby promoting deadenylation. Bic-C overexpression induces premature cytoplasmic-streaming, a posterior-group phenotype, defects in Oskar and Kinesin heavy chain:βGal localization as well as dorsal-appendage defects. These phenotypes are largely reciprocal to those of Bic-C mutants, and they affect cellular processes that Bic-C-associated mRNAs are known, or predicted, to regulate. It is concluded that Bic-C regulates expression of specific germline mRNAs by controlling their poly(A)-tail length (Chicoine, 2007).

Precise coordination of translational control and mRNA localization regulates the temporal and spatial expression of proteins that define the dorsal/ventral and anterior/posterior axes of the Drosophila embryo . These axes are established during oogenesis through the activities of the TGF-α homolog Gurken (Grk) and subsequent posterior accumulation of Oskar (Osk). During oogenesis, osk and grk mRNAs are localized in particular regions of the oocyte cytoplasm, and their localization is dynamic, highly regulated, and essential for their developmental functions. Translation from both of these mRNAs is also under complex regulation (Chicoine, 2007 and references therein).

In wild-type oogenesis, rapid circular streaming of the oocyte cytoplasm begins in late stage 10 and continues until stage 12, when the nurse cells transfer their cytoplasm into the oocyte. Cytoplasmic streaming has been linked to osk localization, because its disruption prevents anterior to posterior translocation of injected osk mRNA in stage-10b to -11 oocytes. Furthermore, Kinesin-1 mutants blocked specifically in cytoplasmic streaming display an abnormal persistence of osk in the center of stage-10 oocytes. Oocytes produced by females homozygous for a hypomorphic orb allele (orbmel) initiate cytoplasmic streaming prematurely. Because orb encodes an RNA-binding protein related to Xenopus cytoplasmic polyadenylation element binding protein (CPEB), this suggests that the timing of this process is regulated through one or more mRNA intermediates (Chicoine, 2007).

Stability and translational activity of maternally transcribed mRNAs are frequently regulated by cytoplasmic proteins that affect their polyadenylation state. In Xenopus oocytes, mos and cyclin B1 mRNAs undergo cytoplasmic elongation of their poly(A) tails at meiotic maturation, and this induces their translation. Cytoplasmic polyadenylation requires CPEB; CPSF (Cleavage and Polyadenylation Specificity Factor) and Symplekin, two factors also involved in nuclear polyadenylation; and Gld2, a cytoplasmic poly(A) polymerase. Cytoplasmic poly(A)-tail elongation depends on phosphorylation of CPEB at meiotic maturation. Before maturation, the polyadenylation complex also contains PARN, a deadenylase whose activity appears to counterbalance Gld2-dependent poly(A)-tail elongation. CPEB phosphorylation leads to a remodeling of the mRNP, which has been proposed to result in the release of PARN from the complex, thus leading to polyadenylation and translational activation (Chicoine, 2007 and references therein).

Regulation of poly(A)-tail length also contributes to regulation of Drosophila mRNAs involved in axis patterning. Orb has been implicated in cytoplasmic polyadenylation of osk mRNA and accumulation of Osk protein at the posterior pole of the oocyte. There is no Drosophila PARN ortholog, and the CCR4-NOT complex, which contains the deadenylase CCR4, POP2, and four NOT proteins, is the major deadenylase in Drosophila (Temme, 2004). The CCR4-NOT deadenylation complex can be recruited to specific mRNA targets in Drosophila embryos, and in yeast, by RNA-binding proteins such as Smaug, Nanos, and PUF-family members, resulting in activated deadenylatio. The mutant phenotypes of twin, the gene encoding CCR4, and measurements of cyclin A and B mRNA poly(A) tails in twin mutants, suggest that regulated deadenylation also occurs in Drosophila oogenesis, but an activator of the CCR4 deadenylase complex in ovaries has not yet been identified (Chicoine, 2007).

Bic-C is required maternally for specifying anterior position during early Drosophila development and for oogenesis (Mohler, 1986; Schüpbach, 1991; Mahone, 1995). Females heterozygous for Bic-C mutations produce embryos of several different phenotypic classes, including bicaudal embryos that consist only of a mirror-image duplication of 2-4 posterior segments. Homozygous Bic-C females are sterile because the centripetal follicle cells fail to migrate over the anterior surface of the oocyte at stage 10. Most egg chambers degenerate shortly after this event. Bic-C protein contains five KH (hnRNP K homology) domains and a SAM (Sterile Alpha Motif) domain. KH and SAM domains are RNA-binding motifs. The KH and SAM domains can also bind to domains of the same type, and SAM domains can bind SH2 domains. Bic-C binds RNA homopolymers in vitro, and a substitution mutation in its third KH domain (G296R) results in substantially decreased RNA-binding activity in vitro and a strong mutant phenotype in vivo. No specific target RNA for Bic-C has heretofore been identified, although Osk accumulation is premature in homozygous Bic-C oocytes (Chicoine, 2007).

This study reports that Bic-C associates with Bic-C mRNA in an ovarian mRNP complex and in gel-shift experiments, and that it can repress its own expression in vivo. Bic-C was overexpressed in germline cells, and premature cytoplasmic streaming, abrogation of posterior osk localization, and dorsal-appendage defects, were observed. The latter phenotype was suppressed, and embryonic viability was increased, by mutations in twin. Furthermore, hiiragi [(hrg) which encodes poly(A) polymerase] and orb mutations are potent dominant enhancers of the Bic-C-overexpression phenotypes. Accordingly, a direct association is found between Bic-C and the NOT3/5 subunit of the CCR4-NOT deadenylation complex, and Bic-C is required for poly(A)-tail shortening of endogenous Bic-C mRNA during early stages of oogenesis. These data show that Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4-NOT deadenylation complex, thus identifying an ovarian activator of this complex. Moreover, the results provide direct evidence in support of the hypothesis that Bic-C and Orb act antagonistically to regulate poly(A)-tail lengths of specific mRNA targets essential for embryonic patterning (Chicoine, 2007).

Several lines of evidence indicate that Bic-C negatively regulates its own expression by binding to an element within its 5'UTR and recruiting the CCR4 deadenylase complex through a direct association with NOT3/5. Several other RNA-binding proteins, such as Nova-1, FMR1P, HuD, PABP, and Orb, bind specifically to their own mRNAs, and, in most cases, these interactions are autoregulatory. Posttranscriptional mechanisms of autoregulation may provide a means of 'fine tuning' levels of regulatory RNA-binding proteins with respect to their target mRNAs, creating the proper equilibrium between silenced and active targets (Chicoine, 2007).

Bic-C-mediated autoregulation is likely essential for development, since overexpression of Bic-C in the female germline induces premature cytoplasmic streaming, which, in turn, produces defects in pole-plasm assembly, posterior patterning, and dorsal-appendage formation. These phenotypes are largely reciprocal to those observed when Bic-C function is reduced, and they are suppressed by reduction of endogenous Bic-C activity. Although the nos::vp16 promoter used to drive UASP-containing transgenes does not recapitulate the normal transcriptional regulation of Bic-C, the level of Bic-C expression it supports is approximately equal to that of wild-type. No attempt was made to overexpress Bic-C from its own promoter, because it was anticipated that doing so in a noninducible manner would result in dominant female sterility resulting from the overexpression phenotypes that were observed. Germline expression of the UASP-Bic-C transgene restored fertility to Bic-CYC33-homozygous females, albeit at a low frequency, possibly due to a lack of fine-tuned regulation, since overexpression phenotypes were observed. This demonstrates that the Bic-C protein produced from UASP-Bic-C is functional. Furthermore, a decrease was observed in the frequency and severity of dorsal-appendage defects induced by Bic-C overexpression through a concomitant reduction of endogenous Bic-C dosage. It is thus likely that the phenotypes observed upon Bic-C overexpression result from an increase in the wild-type function of Bic-C (Chicoine, 2007).

Bic-C-overexpression phenotypes suggest that its targets include mRNAs involved in regulating the onset of rapid cytoplasmic streaming. Consistent with this, overexpression of Bic-CG296R, a form with reduced RNA-binding activity (Saffman, 1998), did not affect cytoplasmic streaming. While the possiblility cannot be excluded that the G296R mutation abrogates other unknown functions of Bic-C, it is noteworthy that several mRNAs that coimmunoprecipated with Bic-C (par-1, Cp190, Cip4, Klp10A, RhoGAP18B, and CG17293) have proven or predicted roles in regulating the actin or tubulin cytoskeleton. It will be important to determine in future experiments whether Bic-C influences cytoplasmic streaming through a regulatory effect on one or more of these potential target mRNAs (Chicoine, 2007).

The results identify Bic-C as an activator of the CCR4 deadenylase complex. Recent data indicate that this complex can be targeted to mRNAs through interactions between different RNA-binding proteins and several of its subunits. PUF proteins interact with the POP2/CAF1 subunit of the complex, as is also likely for Smaug, whereas Nanos binds the NOT4 subunit to recruit the complex to CyclinB 3'UTR. Bic-C directly associates with the NOT3/5 subunit. It is speculated that the ability of different RNA-binding proteins to target different components of the CCR4 complex provides additional regulatory independence and diversity. An uncommon characteristic of activated deadenylation by Bic-C is that binding to the 5'UTR of the regulated mRNA is required, whereas recruitment of the deadenylation complex by other regulatory proteins occurs through 3'UTRs. Circularization of mRNAs through an association between poly(A)-binding protein and eukaryotic initiation factor 4G, which is part of the 5' cap-binding structure, places the 5' and 3'UTRs in close juxtaposition and enables them to function coordinately. Therefore, 3'UTR-binding proteins influence translation initiation. Conversely, this study demonstrates that Bic-C interacts with elements in the 5'UTR and influences poly(A)-tail length. Consistent with this, direct targeting of the yeast CCR4 deadenylation complex to a reporter mRNA results in its rapid decay, regardless of whether the targeting site is in the 3' or 5'UTR of the reporter (Chicoine, 2007 and references therein).

orb mutants produce a premature cytoplasmic-streaming phenotype similar to that of Bic-C overexpression, reduction of orb activity suppresses Bic-C-mutant phenotypes, and this study observed a remarkable enhancement of the Bic-C-overexpression phenotype in a heterozygous orb-mutant background. Because Bic-C overexpression disrupts posterior recruitment of pole-plasm components prior to any detectable effects on Orb levels or distribution, it is concluded that Bic-C and Orb directly regulate the expression of a common set of target mRNAs, rather than Bic-C operating solely through an effect on orb mRNA itself. Consistent with this, Bic-C and Orb proteins have been found in coimmunoprecipitation experiments to be in common mRNP complexes in ovaries. Orb has a role in cytoplasmic polyadenylation of osk mRNA, and genetic interactions suggest that Orb achieves this function together with poly(A) polymerase. Taken together, these data support the model that regulation of the poly(A)-tail length of specific mRNAs results from concomitant polyadenylation and deadenylation regulated by specific RNA-binding proteins. Consistent with this, in Xenopus oocytes, PARN deadenylase is present in the cytoplasmic polyadenylation complex and counteracts polyadenylation prior to meiotic maturation. Both deadenylation and polyadenylation depend on CPEB, the Orb homolog that interacts with both PARN deadenylase and Gld2 poly(A) polymerase. A role in translational repression has not yet been described for Orb, but the observations that Bic-C is involved both in direct activation of deadenylation by CCR4, and also in poly(A)-tail length elongation in later oogenesis, suggests that it is central to poly(A)-tail length regulation and potentially responsible for a switch in the balance between deadenylation and polyadenylation. This switch appears to take place at mid-oogenesis, before stage 9, for Bic-C mRNA, but it could be timed differently for other mRNA targets. The transition from promoting deadenylation to promoting polyadenylation could depend on specific regulatory proteins bound to each mRNA and/or on posttranslational modifications to Bic-C itself (Chicoine, 2007).

The CCR4 Deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal

Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. This study shows that the CCR4 deadenylase is required for GSC self-renewal; Nos and Pum act through its recruitment onto specific mRNAs. mei-P26 mRNA was identified as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. Fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation (Joly, 2013).

This study provides evidence that the twin gene that encodes the CCR4 deadenylase is essential for GSC self-renewal. GSCs are rapidly lost in twin mutants because they differentiate and cannot self-renew. Clonal analysis shows that twin is required cell autonomously in the GSCs for their self-renewal. Nos and Pum are major factors of GSC self-renewal and are translational repressors. Genetic and protein interactions among twin, nos, and pum indicate that CCR4 acts together with Nos and Pum to promote GSC self-renewal. This identifies the recruitment of the CCR4-NOT deadenylation complex as the molecular mechanism underlying Nos and Pum translational repression in the GSCs. Two mechanisms of action used by Nos/Pum have previously been described in the embryo. First, Nos/Pum represses hb mRNA translation by forming a complex with Brat, which in turn interacts with 4EHP and blocks initiation of translation. Second, Nos/Pum represses cyclin B mRNA translation in the primordial germ cells by recruiting the CCR4-NOT complex through direct interactions between Pum and CAF1 and between Nos and NOT4 (Kadyrova, 2007). Brat is not expressed in GSCs, thus excluding the first mode of Nos/Pum translational repression in these cells. However, Pum, Nos, and CCR4 were found to be present in a complex in GSC-like cells, consistent with the recruitment of the CCR4-NOT complex by Nos/Pum for GSC self-renewal (Joly, 2013).

Interestingly, a mutant form of CCR4 that is inactive for deadenylation is able to partially rescue the lack of CCR4 in GSCs. This is consistent with CCR4 not being the only deadenylase in the complex (Temme, 2010). However, CCR4 does participate in the deadenylation activity of the complex, probably via a structural role. Furthermore, the CCR4-NOT complex has been shown recently to be involved in direct translational repression, in addition to its role in deadenylation (Chekulaeva, 2011; Cooke, 2010). This dual mode of action of CCR4-NOT might also be relevant to GSCs (Joly, 2013).

The miRNA pathway also plays a crucial role in GSC self-renewal. A large body of evidence has shown that an important mechanism of silencing by miRNAs involves deadenylation resulting from the recruitment of CCR4-NOT by GW182 bound to Ago1 (for review, see Braun, 2012). Therefore, the CCR4-NOT complex is also likely to contribute to miRNA-mediated translational repression in the GSCs, thus making this complex a central effector of translational repression in the GSCs (Joly, 2013).

An important result from this study is that mei-P26 mRNA is a major target of Nos/Pum/CCR4 regulation for GSC self-renewal. Nos and Pum are known to be essential players in GSC self-renewal, and many mRNAs are expected to be regulated by this complex. However, to date only one mRNA target of this complex, brat, has been reported. This study has identified another target, mei-P26 mRNA, and has shown that its repression by the Nos/Pum/CCR4 complex has a key role in GSC self-renewal, because the loss of GSCs in the twin mutant is strongly rescued by decreasing mei-P26 gene dosage (Joly, 2013).

Both Brat and Mei-P26 belong to the Trim-NHL family of proteins, which have conserved functions in stem cell lineages from C. elegans to mouse (for review, see Wulczyn, 2010). Proteins within this family are potential E3 ubiquitin ligases and can act by either activating or antagonizing the miRNA pathway, through their association with Ago1 and GW182. In particular, Mei-P26 function switches from activation of the miRNA pathway in the GSCs to inhibition of the pathway in differentiating cysts where Mei-P26 levels are higher. As such, Mei-P26 plays a central role in the control of cell fate in the GSC lineage. The rescue of the twin mutant phenotype of GSC loss by decreasing mei-P26 gene dosage suggests that the levels of Mei-P26 themselves might be important for this switch of its function. This might provide an explanation as to why such a precise regulation of its level is crucial for GSC self-renewal and differentiation (Joly, 2013).

Which molecular mechanisms underlie the fine-tuning of Mei-P26 in the GSC lineage? The translational repression of mei-P26 mRNA is not complete in GSCs. This differs from the complete repression by Nos/Pum of cyclin B mRNA in the primordial germ cells, or brat mRNA in the GSCs, and may result from the concomitant activation of mei-P26 by Vasa. Vasa does activate mei-P26 translation, leading to a peak of expression in 8-cell and 16-cell cysts. However, Vasa is expressed in all germ cells, suggesting that it is not the key regulator governing the timing of Mei-P26 peak of expression. It is proposed that translational activation of Mei-P26 by Vasa would be active already in GSCs but counterbalanced by translational repression by Nos/Pum and the CCR4-NOT complex. In cystoblasts, the presence of Bam overcomes Nos/Pum translational repression by decreasing Nos levels, which would thus switch the balance to translational activation by Vasa. This does not lead to a peak of Mei-P26 expression in cystoblasts, but rather to a progressive increase of Mei-P26 levels in proliferating cysts. This progressive accumulation of Mei-P26 could depend on the necessity to build up Vasa-mediated translational activation. However, another possibility could be that a different factor still partially represses mei-P26 translation in cystoblasts and early cysts. A potential candidate is Bam, which has been defined as a translational repressor and has recently been reported to directly repress mei-P26 mRNA translation in the male GSC lineage (Insco, 2012). The Bam expression profile in female germ cells is consistent with this potential role in mei-P26 translational repression, because Bam protein is present from cystoblasts to 8-cell cysts but absent in 16-cell cysts, where Mei-P26 levels are the highest (Joly, 2013).

Recent advances have established the generality of a central role for translational regulations in adult stem cell lineages. Translational repression is required to prevent the synthesis of differentiation factors whose mRNAs are already present in stem cells. In the Drosophila female GSC lineage, recent work has demonstrated that changes in cell fate are driven by different translational regulation programs; associations between translational repressors evolve to trigger stage-specific regulation of mRNA targets. For example, while Nos/Pum maintain female GSCs by repressing a specific set of mRNAs, Pum associates with Brat in cystoblasts to repress a different set. The Trim-NHL proteins appear to be of particular importance in the translational regulations essential for stem cell fate as exemplified by Mei-P26. The fine-tuning of Mei-P26 protein levels by translational repression is essential for GSC self-renewal and implicate CCR4 in this regulation (Joly, 2013).

The functions of Trim-NHL proteins are conserved in many adult stem cell lineages in different organisms, and mutations in the corresponding genes lead to highly proliferative tumors. Elucidating the molecular mechanisms behind their translational control is key to deciphering how these proteins regulate adult stem cell fates (Joly, 2013).

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. This study demonstrates that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. These findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation (Zekri, 2013).

HPat a decapping activator interacting with the miRNA effector complex

Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study split-affinity purifications was performed in Drosophila cells and evidence is provided for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors the importance of NOT1 for this interaction was demonstrated. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation (Barisic-Jager, 2013).

Distinct modes of recruitment of the CCR4-NOT complex by Drosophila and vertebrate Nanos. EMBO J 35(9):974-90. PubMed ID: 26968986

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non-conserved N-terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4-NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4-NOT deadenylase complex (see Drosophila Twin) in invertebrates. This study identified an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4-NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C-terminal domains of NOT1-3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N-terminal regions of Nanos proteins recruit CCR4-NOT to assemble analogous repressive complexes (Raisch, 2016).

Post-transcriptional mRNA regulation plays an essential role in embryonic development. This regulation is mediated by RNA-binding proteins that control the spatial and temporal expression of target mRNAs through the recruitment of effector complexes. The RNA-binding proteins of the Nanos family are conserved post-transcriptional mRNA regulators that play essential roles in embryonic germline specification, germline stem cell maintenance, and neuronal homeostasis in Drosophila melanogaster (Dm) and a wide range of other. The Dm Nanos protein is also required for posterior pattern formation in the embryo (Raisch, 2016).

Three Nanos paralogs (Nanos1-3) exist in vertebrates and various invertebrate species, whereas there is only one family member in Dm and other insects. This protein family is defined by a highly conserved CCHC-type zinc-finger (ZnF) domain and divergent N- and C-terminal unstructured regions of variable lengths. The ZnF domain mediates binding to RNA and to Pumilio, a conserved Nanos partner that confers mRNA target specificity. The unstructured regions are required for interaction with effector complexes, which include the CCR4-NOT deadenylase complex embryo (Raisch, 2016).

The CCR4-NOT complex catalyzes the removal of mRNA poly(A) tails and consequently represses translation. In addition, dead-enylation by the CCR4-NOT complex is coupled to decapping and 5'-to-3' exonucleolytic degradation by XRN1 and can therefore lead to full mRNA degradation in some cellular contexts. Furthermore, the CCR4-NOT complex can also repress translation independently of deadenylation (Raisch, 2016).

The CCR4-NOT complex consists of several independent modules that dock with NOT1, a central scaffold subunit (Temme, 2014). NOT1 consists of independently folded α-helical domains that provide binding sites for the individual modules. A central domain of NOT1, structurally related to the middle domain of eIF4G (the NOT1 MIF4G domain), provides a binding site for the catalytic module, which comprises two deadenylases, namely CAF1 (or its paralog POP2) and CCR4a (or its paralog CCR4b) (Raisch, 2016).

The C-terminal region of NOT1 contains the NOT1 superfamily homology domain (SHD) and assembles with NOT2-NOT3 heterodimers to form the NOT module. The NOT module provides binding sites for RNA-binding proteins, such as vertebrate Nanos and Dm Bicaudal-C, which recruit the CCR4-NOT complex to their mRNA targets (Raisch, 2016).

The three vertebrate Nanos paralogs contain a 17-amino acid NOT1-interacting motif (NIM) that binds directly to the NOT1 SHD domain. Although the NIM is conserved in vertebrate Nanos, Nanos proteins of insects and worms do not have a detectable NIM. Nevertheless, Dm Nanos has been reported to interact with NOT4 through its unstructured N-terminus. However, because NOT4 is not stably associated with the CCR4-NOT complex in metazoans, it has remained unclear whether Dm Nanos recruits the CCR4-NOT complex to mRNA targets directly or rather relies on its interaction with additional partners, such as Pumilio (PUM) and Brain tumor (BRAT), to exert its repressive function (Raisch, 2016).

This study shows that although Dm Nanos does not contain a NIM, it interacts directly with the CCR4-NOT complex using an extended region that is termed the Nanos effector domain (NED). The NED overlaps with a region previously shown to contribute to Nanos function in Dm embryos. The crystal structure of a central region of the NED (termed the NOT module binding region, NBR) bound to the NOT module revealed a bipartite interface that contacts both the NOT1 SHD and the NOT3 NOT-box domains. The binding site for the Dm NBR on NOT1 does not overlap with the vertebrate NIM-binding site. These results indicate that Nanos proteins have maintained the ability to interact with the CCR4-NOT complex using divergent motifs in disordered protein regions (Raisch, 2016).

This study has shown that Dm Nanos recruits the CCR4-NOT complex directly through a Nanos effector domain (NED) that is conserved in the Drosophila species. Similar to the vertebrate Nanos NIM, the Dm NED is necessary and sufficient to repress translation in the absence of mRNA degradation and to promote degradation of bound mRNAs. Thus, the NED and the NIM are the main determinants for the repressive activity of Nanos in Dm and vertebrates, respectively. Although the NED and the NIM are functionally analogous, they do not share sequence similarities, demonstrating that the absence of sequence conservation is not an indicator of functional irrelevance, in particular, when disordered, low complexity protein regions are involved. Such regions often mediate their function by interacting with binding partners using short linear motifs (SLiMs). SLiMs can evolve rapidly due to the lack of constraints to maintain a protein fold and thus enable the evolution of distinct binding modes in orthologous proteins, especially in cases where these proteins are in a competitive scenario or even under positive selection. In this way, these proteins can maintain the ability to interact with the same partners using different binding modes (Raisch, 2016).

The Dm NED and the vertebrate NIM use different modes to mediate the recruitment of the CCR4-NOT complex to Nanos mRNA targets. The NIM is a short 17-residue motif present in the N-terminal disordered region of vertebrate Nanos proteins, which binds to the NOT1 SHD domain. In contrast to vertebrates, flies have only a single Nanos protein but with an extended NED. The Dm NED is 187 amino acids in length and contains multiple and redundant binding sites for the CCR4-NOT complex. These multiple binding sites may increase the affinity of Dm Nanos for the CCR4-NOT complex through avidity effects. Redundancy may also confer a competitive advantage to Dm Nanos over other RNA-binding proteins that compete for recruitment of the CCR4-NOT complex (Raisch, 2016).

Interestingly, redundancy to recruit the CCR4-NOT complex is not only observed within the Nanos protein but also in the context of Nanos repressive complexes. Indeed, Nanos cooperates with PUM to bind and repress natural mRNA targets, and PUM also has the ability to recruit the CCR4-NOT complex independently of Nanos. This may partially obscure the effects of deletion or mutations in the Nanos NED. However, PUM does not act as a general substitute for Nanos because mutations in the Nanos ZnF domain that prevent mRNA binding caused strong developmental defects despite the presence of endogenous PUM. Thus, PUM probably acts both additively and alternatively with the Nanos NED, resulting in distinct modes of engaging the CCR4-NOT complex. In their various combinations, the different binding modes can thus lead to a highly specific and tunable repression of mRNA targets in a cell context-dependent manner (Raisch, 2016).

A large number of RNA-associated proteins have been shown to recruit the CCR4-NOT complex to their mRNA targets to repress translation and/or to promote mRNA degradation. In addition to Nanos, these proteins include the GW182 proteins, which are involved in miRNA-mediated gene silencing in animals, and the Dm proteins CUP, Bicaudal-C, Smaug, and PUM. Additional examples from vertebrates are Roquin and tristetraprolin (TTP), a protein required for the degradation of mRNAs containing AU-rich elements (ARE-mediated mRNA decay) (Raisch, 2016).

For the recruitment of the CCR4-NOT complex, most of these proteins rely on short linear motifs (SLiMs) embedded in peptide regions of predicted disorder. However, a detailed characterization of their interaction with the CCR4-NOT complex on a molecular level is only available for TTP, GW182, and vertebrate and Dm Nanos. For TTP and vertebrate and Dm Nanos, the motifs adopt α-helical conformations that possibly form only upon binding. Specificity results from aromatic and hydrophobic side chains that insert primarily into pockets on the surface of the NOT1 domains that consist of HEAT-like repeats. By contrast, GW182 peptides likely bind to the CCR4-NOT complex in an extended conformation and insert tryptophan residues into tandem hydrophobic pockets exposed at the surface of the NOT9 subunit (also known as CAF40) of the CCR4-NOT complex and probably into additional pockets in NOT1 that remain to be identified (Raisch, 2016).

Similar to GW182 proteins, the Nanos NBR not only contacts NOT1 but also binds to NOT3, providing the first detailed insight into how an mRNA-binding protein recruits the CCR4-NOT complex by contacting two of its subunits simultaneously. Interestingly, the surface on NOT1 that is contacted by Dm Nanos partially overlaps with the binding surface for NOT4 as observed in the Saccharomyces cerevisiae (Sc) complex. This would suggest that Dm Nanos competes with NOT4 for binding to NOT1, providing additional opportunities for the regulation of gene expression. However, it is not known whether the NOT4 binding mode is conserved between Dm and Sc because NOT4 does not co-purify with the CCR4-NOT complex in metazoans and the Sc NOT4 sequences that bind NOT1 are not well conserved in metazoans (Raisch, 2016).

Together with previous studies (Fabian, 2013; Bhandari, 2014; Chen, 2014; Mathys, 2014), these results reveal that the recruitment of the CCR4-NOT complex is mediated by highly diverse sequence motifs and distinct binding modes. It is speculated that these motifs represent a combinatorial code that is read by the CCR4-NOT complex to funnel the effects of diverse RNA-binding proteins into a common repressive pathway, which results in the removal of the mRNA poly(A) tail, translational repression, and, depending on the cellular context, full degradation of the mRNA (Raisch, 2016).


Search PubMed for articles about Drosophila CCR4

Albert, T. K., et al. (2000). Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits. Nucleic Acids Res. 28: 809-817. PubMed ID: 10637334

Baggs, J. E. and Green, C. B. (2003). Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA. Curr Biol 13: 189-198. PubMed ID: 12573214

Barisic-Jager, E., Krecioch, I., Hosiner, S., Antic, S. and Dorner, S. (2013). HPat a decapping activator interacting with the miRNA effector complex. PLoS One 8: e71860. PubMed ID: 23977167

Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P. and Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20(14): 1885-98. PubMed ID: 16815998

Bhandari, D., Raisch, T., Weichenrieder, O., Jonas, S., Izaurralde, E. (2014). Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression. Genes Dev. 28(8):888-901. PubMed ID: 24736845

Boeck, R., et al. (1996). The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity. J. Biol. Chem. 271: 432-438. PubMed ID: 8550599

Braun, J. E., Huntzinger, E. and Izaurralde, E. (2012). A molecular link between miRISCs and deadenylases provides new insight into the mechanism of gene silencing by microRNAs. Cold Spring Harb Perspect Biol 4. PubMed ID: 23209154

Brown, C. E., Tarun, S. Z., Boeck, R. and Sachs, A. B. (1996). PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae. Mol Cell Biol. 16: 5744-5753. PubMed ID: 8816488

Brown, C. E. and Sachs, A. B. (1998). Poly(A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Mol Cell Biol 18: 6548-6559. PubMed ID: 9774670

Castagnetti, S. and Ephrussi, A. (2003). Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte. Development 130: 835-843. PubMed ID: 12538512

Chen, Y., Boland, A., Kuzuoglu-Õzt├╝rk, D., Bawankar, P., Loh, B., Chang, C. T., Weichenrieder, O., Izaurralde, E. (2014). A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing. Mol Cell. 54(5):737-50. PubMed ID: 24768540

Chicoine, J., Benoit, P., Gamberi, C., Paliouras, M., Simonelig, M. and Lasko, P. (2007). Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression. Dev. Cell 13(5): 691-704. PubMed ID: 17981137

Chekulaeva, M., Mathys, H., Zipprich, J. T., Attig, J., Colic, M., Parker, R. and Filipowicz, W. (2011). miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs. Nat Struct Mol Biol 18: 1218-1226. PubMed ID: 21984184

Chen, J., et al. (2001). Purification and characterization of the 1.0 MDa CCR4-NOT complex identifies two novel components of the complex. J. Mol. Biol. 314: 683-694. PubMed ID: 11733989

Chen, J., Chiang, Y. and Denis, C. L. (2002). CCR4, a 3'-5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase. EMBO J 21: 1414-1426. PubMed ID: 11889047

Collart, M. (2003). Global control of gene expression in yeast by the Ccr4-Not complex. Gene 313: 1-16. PubMed ID: 12957374

Cooke, A., Prigge, A. and Wickens, M. (2010). Translational repression by deadenylases. J Biol Chem 285: 28506-28513. PubMed ID: 20634287

Cougot, N., Babajko, S. and Seraphin, B. (2004). Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165: 31-40. PubMed ID: 15067023

Couttet, P., et al. (1997). Messenger RNA deadenylation precedes decapping in mammalian cells. Proc. Nat. Acad. Sci. 94: 5628-5633. PubMed ID: 9159123

Daugeron, M.-C., Mauxion, F. and Seraphin, B. (2001). The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Res. 29: 2448-2455. PubMed ID: 11410650

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Biological Overview

date revised: 22 March 2022

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