InteractiveFly: GeneBrief

pacman: Biological Overview | References

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. This study shows that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. These results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing (Nishioka, 2018).

Polycomb silencing is essential for the developmental regulation of gene expression. The silencing needs to be robust to tightly repress the expression of developmental genes in undifferentiated cells, such as stem cells, but should also be flexible for rapid release upon differentiation. However, this paradoxical aspect of Polycomb silencing is not well understood (Nishioka, 2018).

Mbf1 was originally identified as an evolutionarily conserved coactivator that connects a transcriptional activator with the TATA element-binding protein (Li, 1994; Takemaru, 1997; Takemaru, 1998). Usually, Mbf1 is present in the cytoplasm; however, under stress conditions, Mbf1 translocates into the nucleus to induce stress-response genes. Previous studies have revealed roles for the coactivator in axon guidance, oxidative stress response, defense against microbial infection, and resistance to drugs such as tamoxifen. However, the cytoplasmic role of Mbf1 has remained elusive, except for mRNA or ribosomal binding (Nishioka, 2018).

Pacman (Pcm/Xrn1) is an evolutionarily conserved 5'-3' exoribonuclease that degrades decapped mRNA (Till, 1998; Jones, 2012). Genetic studies have demonstrated that Drosophila pcm is involved in epithelial closure, male fertility, apoptosis and growth control (Grima, 2008; Lim, 2009; Jones, 2012; Jones 2016; Waldron, 2015). Null mutants of pcm are lethal during early pupal stages, suggesting the enzyme plays an essential role in development (Waldron, 2015; Jones, 2016; Nishioka, 2018 and references therein).

Using a genetic approach in Drosophila, this study shows that cytoplasmic Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pcm. The results thus demonstrate an unexpected component of the regulatory mechanism underlying Polycomb silencing. This mechanism might also allow flexibility in Polycomb silencing, as Mbf1 protein expression declines upon differentiation (Nishioka, 2018).

To address the cytoplasmic role of Mbf1, novel genes were sought that interact with mbf1. Surprisingly, the mbf1 mutation enhanced a classical Polycomb phenotype of Psc and Pc mutants, namely the appearance of an ectopic sex comb tooth or teeth on the male mid-leg. Although mbf1²/+ or mbf1²/mbf1² flies never exhibited the Polycomb phenotype, penetrance of the phenotype in Psc¹/+ increased significantly in Psc¹/+; mbf1²/+, and further increased in Psc¹/+; mbf1²/mbf1². The penetrance was restored to the Psc¹/+ level by expressing wild-type Mbf1 protein from a transgene. Similar effects of the mbf1² allele were observed with the Pc⁶ mutation (Nishioka, 2018).

To gain insight into the mechanism underlying the genetic interaction between Psc and mbf1, the expression of the representative Polycomb group genes Pc, E(z) and pho was analyzed. Results of reverse transcription-quantitative PCR (RT-qPCR) analyses demonstrated a prominent reduction in the expression level of E(z) mRNA in Psc¹/+; mbf1²/+ larvae, whereas Pc and pho mRNA levels remained unchanged. Immunostaining of wing discs demonstrated that E(z) protein expression was severely compromised in Psc¹/+; mbf1²/+ compared with that in wild type, mbf1²/+ or Psc¹/+. By contrast, the expression of Pc and Pho proteins was not significantly affected. Western blot analyses confirmed the marked decrease in the E(z) protein level in both wing and leg discs from Psc¹/+; mbf1²/+. Consistently, Psc¹/+; E(z)⁷³¹/+ exhibited the extra sex comb phenotype, which was comparable to Psc¹/+; mbf1²/+ (Nishioka, 2018).

It is unlikely that Mbf1 affects E(z) transcription because no significant difference was detected in the E(z) mRNA level between wild-type and mbf1²/mbf1² larvae. Consistently, it was not possible to detect any significant difference in the expression of E(z) in the wing disc upon knockdown or overexpression of Mbf1 using a posterior compartment-specific Gal4 driver. When cytoplasmic and nuclear RNA fractions from wing discs were analyzed by RT-qPCR, the nuclear E(z) mRNA level was similar between wild type and Psc¹/+; mbf1²/+. However, the cytoplasmic E(z) mRNA level in Psc¹/+; mbf1²/+ decreased to ~20% of the wild-type level. Collectively, these results suggest that mbf1 regulates the E(z) mRNA level post-transcriptionally in the cytoplasm (Nishioka, 2018).

Considering that Mbf1 binds to mRNA, it was hypothesized that cytoplasmic Mbf1 might bind to E(z) mRNA to protect it from degradation, and thereby regulates the E(z) mRNA level. Results of RNA-immunoprecipitation (RIP) experiments revealed a preferential binding of Mbf1 to E(z) mRNA. A ~10-fold enrichment of E(z) mRNA was found in the anti-Mbf1 antibody pull-down fraction from cytoplasmic extracts of embryos. The pull-down was clearly selective, as enrichment of abundant mRNAs, such as RpL32 and RpL30, was not observed. By contrast, E(z) mRNA was barely detectable in the anti-Mbf1 antibody pull-down fraction from embryonic extracts of the mbf1 mutant, used as a negative control. This is not due to absence of E(z) mRNA in the mbf1 mutant (Nishioka, 2018).

Following the observed preferential binding of Mbf1 to E(z) mRNA, this study focused on the Polycomb phenotype and reduced E(z) mRNA expression level, which were not caused by the mbf1 mutation alone. Enhancement of the Polycomb phenotype and the reduction of E(z) mRNA were only detected in the double mbf1 and Polycomb group gene mutant. To explain the synergistic effect of mbf1 and Polycomb group mutations, it was posited that a component of the mRNA degradation pathway was only activated in the Polycomb group mutant background. Therefore, attempts were made to identify the component of the pathway that was activated in the Psc or Pc mutants. Among the mRNAs tested, only pcm mRNA, which encodes the 5'-exoribonuclease, was upregulated in Psc¹/+ and Pc⁶/+ larvae. Neither the decapping enzyme (Dcp2), components of the exosome [Dis3, Prp6 (CG6841) and Prp40 (CG3542)], nor components in the 3'-deadenylation-mediated pathway (twin and Nab2) appeared to be activated. Western blot analyses revealed a 2-fold increase in the Pcm protein level in wing discs from Psc¹/+ or Pc⁶/+ larvae compared with that from wild type. These results led to an investigation of the effects of the pcm mutation on Polycomb silencing and E(z) mRNA expression (Nishioka, 2018).

Strikingly, the pcm^Δ1 mutation resulted in significant suppression of the Polycomb phenotype in Psc¹/+ and Psc¹/+; mbf1²/+. This suppression was rescued by expressing the wild-type Pcm protein from a transgene. Similar results were obtained using the Pc⁶ mutant. Consistent with this result, the pcm^Δ1 mutation restored the E(z) mRNA levels in Psc¹/+ and Psc¹/+; mbf1²/+ to near wild-type levels (Nishioka, 2018).

In addition to the extra sex comb phenotype, Psc¹/+; mbf1²/+ exhibited misexpression of Ubx in wing discs. The signals appeared as spots consisting of clusters of Ubx-positive cells. The pcm^Δ1 mutation decreased the number of spots per wing disc. The misexpression occurred predominantly around the dorsoventral border in the posterior compartment. Consistently, adult wing defects were observed along the posterior wing margin, which was also suppressed by pcm^Δ1 (Nishioka, 2018).

Importantly, the extra sex comb phenotype was detected under mild overexpression of pcm in mbf1²/hs-pcm double heterozygotes at 25°C, even in the wild-type Polycomb group background. hs-pcm/+ exhibited an ~2.5-fold overexpression of Pcm at 25°C. Nevertheless, hs-pcm heterozygotes in the wild-type mbf1 background did not show any Polycomb phenotype. These results suggest that Mbf1 stabilizes Polycomb silencing against fluctuations in the Pcm protein level in vivo. Enhancement of the Polycomb phenotype was also observed in Psc¹/+; hs-pcm/+ compared with that in Psc¹/+ (Nishioka, 2018).

Biochemical analyses using purified recombinant Mbf1 and Pcm proteins revealed that Mbf1 protects E(z) RNA from degradation by Pcm. RNA protection assays were performed in which in vitro-synthesized E(z) RNA was treated with the RNA pyrophosphatase RppH to convert the 5'-triphosphoryl end into the 5'-monophosphoryl form, which is a Pcm substrate. The RNA was digested with Pcm in the presence or absence of Mbf1. Mbf1 inhibited the digestion of E(z) RNA. In the absence of RppH, RNA degradation was barely detectable, suggesting that the digestion was due to 5'-exoribonuclease activity. Gel filtration of a mixture of Pcm and Mbf1 resulted in the elution of each protein in a clearly separated peak. Furthermore, Mbf1 did not co-immunoprecipitate with Pcm and vice versa. These results suggest that Mbf1 does not inhibit Pcm activity through protein-protein interactions. Collectively, it is concluded that Mbf1 protects E(z) mRNA from degradation by Pcm both in vivo and in vitro (Nishioka, 2018).

It is proposed that cytoplasmic Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from the activity of Pcm. In the mbf1 mutant, E(z) mRNA is free from Mbf1 protein, but pcm expression is downregulated by Polycomb group genes. In the Polycomb group mutant, Pcm expression is upregulated, but E(z) mRNA is partly protected by Mbf1. In the mbf1 Polycomb group double mutant, E(z) mRNA is free from Mbf1 protein and is subject to Pcm attack. Whereas Mbf1 is highly expressed in undifferentiated cells, such as those of embryos, larval testis, ovary, imaginal discs and neuroblasts, its expression is reduced in differentiated tissues, similar to the situation in the mbf1 mutant. This would facilitate the rapid release of developmental genes from Polycomb silencing upon differentiation. Interestingly, expression of mammalian Mbf1 [also termed endothelial differentiation-related factor 1 (Edf1)] and Ezh2 declines immediately after the onset of differentiation (Nishioka, 2018).

A recent study demonstrated that Pcm prevents apoptosis in imaginal discs and downregulates specific transcripts such as hid and reaper (Waldron, 2015). However, suppression of apoptosis did not rescue the lethality of a pcm null mutation at the early pupal stage. Therefore, there might be other targets of Pcm that are essential for early pupal development. The present study indicates that E(z) mRNA could be one such target (Nishioka, 2018).

The mRNA-binding activity of Mbf1 was selective, but might not be strictly specific to E(z) mRNA. Although Polycomb silencing is central to the developmental regulation of gene expression, there could be other mRNAs that bind to Mbf1 in a similar manner, thereby modulating another biological function. Therefore, RIP-seq analysis was conducted to identify Mbf1-bound mRNAs. To ensure robustness of the RIP-seq data, the results were compared independently with two publically available datasets and identified 804 commonly enriched mRNAs. Among these, the enrichment of four representative mRNAs (GstD5, Ide, Tep2 and Pebp1) was confirmed by RIP RT-qPCR analyses. Interestingly, the expression levels of these four mRNAs decreased in Psc¹/+; mbf1²/+ and increased in pcm^Δ1/Y compared with those in wild type, suggesting that the model can be applied to a wider range of mRNAs than just E(z). However, dependency on the Mbf1/Pcm antagonism appears to differ among the mRNAs (Nishioka, 2018).

Gene ontology and pathway analyses of the 804 genes revealed some interesting properties of the Mbf1-associated mRNAs. The gene ontology terms 'glutathione metabolic process', 'oxidation-reduction process' and 'neurogenesis' which includes E(z), are consistent with the fact that previous studies found defects in oxidative stress defense and axon guidance in the mbf1 mutant (Liu, 2003; Jindra, 2004). Also of interest are the groups 'positive regulation of innate immune response' and 'defense response to Gram-negative bacterium', as Arabidopsis MBF1 is involved in host defense against microbial infection. Moreover, pathway analysis of the enriched genes implicated Mbf1 in 'drug metabolism', as previously suggested for tamoxifen resistance. This raises an intriguing possibility that Mbf1 contributes to various types of stress defense, metabolic processes and neurogenesis as both a nuclear coactivator and as a cytoplasmic mRNA-stabilizing protein. Although mbf1 null mutants are viable under laboratory conditions, evolutionary conservation of mbf1 suggests that it has essential role(s) under real-world stress conditions (Nishioka, 2018).

RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1

Ribonucleases are critically important in many cellular and developmental processes and defects in their expression are associated with human disease. Pacman/XRN1 is a highly conserved cytoplasmic exoribonuclease which degrades RNAs in a 5'-3' direction. In Drosophila, null mutations in pacman result in small imaginal discs, a delay in onset of pupariation and lethality during the early pupal stage. This paper has have used RNA-seq in a genome-wide search for mRNAs misregulated in pacman null wing imaginal discs. Only 4.2% of genes are misregulated ±>2-fold in pacman null mutants compared to controls, in line with previous work showing that Pacman has specificity for particular mRNAs. Further analysis of the most upregulated mRNAs showed that Pacman post-transcriptionally regulates the expression of the secreted insulin-like peptide Dilp8. Dilp8 is related to human IGF-1, and has been shown to coordinate tissue growth with developmental timing in Drosophila. The increased expression of Dilp8 is consistent with the developmental delay seen in pacman null mutants. This analysis, together with previous results, show that the normal role of this exoribonuclease in imaginal discs is to suppress the expression of transcripts that are crucial in apoptosis and growth control during normal development (Jones, 2016).

This study used RNA-seq in a global approach to identify candidate targets of the exoribonuclease Pacman during imaginal disc development. Using two pacman null mutants, together with their respective wild-type controls, two new potential targets of Pacman, dilp8 and Nplp2, were identified. These are upregulated 6600-fold and 38-fold respectively at the post-transcriptional level, as assessed by qRT-PCR. Dilp8 is a secreted insulin-like peptide which is known to co-ordinate tissue growth with developmental timing. The upregulation of Dilp8 protein, which was visualized in L3 imaginal discs, is consistent with the developmental delay seen in the pacman null homozygous larvae. Nplp2 (neuropeptide-like precursor-2) transcripts are predicted to encode neuropeptides of 86 and 119 amino acids, the functions of which are unknown. this analysis, together with previous published results, suggests that transcripts encoding short peptides are particularly sensitive to regulation by Pacman. They also suggest that the role of this exoribonuclease is to suppress the expression of transcripts that are crucial in apoptosis and in endocrine signalling during normal development (Jones, 2016).

The data highlights the importance of tightly controlled experiments using two null mutants, together with appropriate wild-type controls to improve the accuracy of the RNA-seq results and account for any differences in genetic background. Ther RNA-seq results show that more genes are significantly upregulated >2-fold (488) than downregulated >2-fold (188) which is consistent with the exoribonuclease function of Pacman. A novel 'inconsistency index' was applied which allows the consistency of replicates to be expressed as a value between 0 and 1 without being biased by FPKM or expression changes between conditions, to narrow down the subset for validation by qRT-PCR. For those genes that are significantly upregulated >2-fold in the pcm¹⁴ mutant by RNA-seq, the median locus size is 4306 bp (range 282-138 435 bp) whereas for genes significantly downregulated >2-fold, the median locus size is 6290 bp (range 214-202 218 bp). This suggests that Pacman tends to target shorter transcripts, though it should be noted that these results include both direct and indirect targets of Pacman and the proportion of each type is currently unknown. Although the GO enrichment scores are not high, they are consistent with the phenotypes observed in pacman mutants and suggest that Pacman affects a number of annotated cellular pathways in Drosophila (Jones, 2016).

The data is consistent with upregulated RNAs being translated and functional as Dilp8 protein is expressed in pcm¹⁴ wing imaginal discs. Therefore the dilp8 transcripts upregulated in the pcm¹⁴ mutants would appear to be capped and polyadenylated. This fits well with previous studies, where the family of Pacman/Xrn1 have been shown to include short linear motifs within their less structured C-terminal regions which bind co-factors involved in 5'-3' degradation. In the case of Drosophila Pacman, the decapping activator Dcp1 binds a DCP1-binding motif within the C-terminal of the protein, thus linking decapping with 5'-3' degradation. In a Pacman mutant, decapping would be expected to be severely inhibited resulting in upregulated transcripts which can be translated (Jones, 2016).

The results described in this paper are also in agreement with previous publications using genetic approaches to analyse the effects of the pcm¹⁴ mutant (Waldron, 2015) and microarray analysis to identify the potential Pacman targets using pcm⁵, a hypomorphic allele (Jones, 2013). reaper is upregulated by 4.3-fold in the pcm¹⁴ mutant by RNA-seq, which is in line with the 7.6-fold change in expression previously quantified by qRT-PCR. A comparison between the RNA-seq results and previous microarray experiments performed on pcm⁵ is shown. These results show a similar upregulation for 10 mRNAs but no upregulation for Dilp8 or reaper. However, relatively few genes changed significantly on the microarrays and the reason for the difference between these datasets is most likely to be both developmental and biochemical. Pacman is ~66.6% functional in the pcm⁵ mutant and the wing imaginal discs are 81.7% the size of wild-type, allowing the pupae to develop into adults with smaller wings without a delay before pupariation. However, the imaginal discs in the pcm¹⁴ null mutant are only 45.0% the size of wild-type, pupariation is significantly delayed and this mutation is completely lethal. It is therefore not surprising that the genes upregulated in the pcm⁵ mutant represent only a subset of those upregulated in the pcm¹⁴ mutant. It is highly likely that dilp8, reaper and other mRNAs are only upregulated once a Pacman function is reduced past a critical threshold. In addition, it is possible that the technical differences between microarray and RNA-seq could give rise to variability between these datasets (Jones, 2016).

Previous reports using the single cell organism Saccharomyces cerevisiae have shown that Xrn1 may buffer mRNA levels by entering the nucleus to interact with transcriptional repressors, thus reducing transcription. No evidence was seen for this for Pacman in the multicellular organism Drosophila. In this and previous publications, wTaqMan assays were used designed to distinguish expression changes at the post-transcriptional level from changes at the transcriptional level. Therefore it can be confidently stated that the RNAs observed to be post-transcriptionally upregulated are not upregulated at the transcriptional level. Furthermore, Pacman was never observed to be present in the nuclei of Drosophila cells. In any case, the experiments aimed to identify the transcripts whose overall expression is substantially perturbed in the pcm¹⁴ mutant as these transcripts are more likely to result in the biological phenotypes observed. If there is a mechanism to maintain homeostasis for some transcripts then these transcripts are unlikely to cause the mutant phenotypes (Jones, 2016).

The finding that dilp8 transcripts are highly upregulated in pacman null mutants is concordant with a previous results showing that these pacman mutations lead to a developmental delay in pupariation. The Drosophila insulin-like peptide family comprise eight members (Dilp1-Dilp8) which are secreted peptides required in development, growth, metabolism, stress responses and lifespan. These peptides display structural similarities to mammalian insulins, insulin-like growth factors and relaxins, including well-conserved positions of cysteines and disulfide bridges. One of the most studied peptides is Dilp2, which is produced by insulin producing cells in the brain of Drosophila and regulates developmental timing, body weight, lifespan, fecundity and trehalose levels, whereas Dilp6 plays a role in reallocating energy stores during pupation. Dilp8 has previously been reported to be highly induced in response to cellular damage (e.g. by gamma-ray irradiation) and in conditions where growth impairment produces a developmental delay. In regenerating tissues, Dilp8 is known to be secreted in vesicle-like structures from the imaginal discs and remotely acts on the brain complex to suppress ecdysone production and activity and therefore delay the onset of pupariation. Previous publications have shown that tissue specific overexpression of Dilp8 results in developmental delay. For example, overexpression of dilp8 using the tubulin promoter results in a pupariation delay of 55.9 h, whereas ectopic expression of dilp8 using the disc specific rotund promoter delays pupariation by 2-3 days. These results are in line with the 32 h delay seen in pcm¹⁴ mutants (Jones, 2016).

The results suggest that the normal function of Pacman in L3 wing imaginal discs is to repress the expression of dilp8, so that the growth status of this tissue is coordinated with developmental timing. The effect of Pacman on dilp8 transcripts in Drosophila is specific as dilp6, the only other member of the Dilp family expressed in wing imaginal discs, is unchanged at the RNA level in the mutant discs compared to the wild-type. Since Pacman (XRN1) is highly conserved in humans and insulin-like peptides are also involved in tissue growth and homeostasis, it is possible that XRN1 regulates similar processes in humans (Jones, 2016).

Previous work on Dilp8 has shown that it is part of a gene regulatory network coordinating abnormal growth of a tissue to the overall growth programme. An increase in levels of dilp8 in imaginal discs has previously been shown to be correlated with increased expression of thor mRNA. Thor (4E-BP) is known to bind to the cap-binding protein 4E and inhibit the initiation of protein translation which is consistent with slower imaginal disc growth. However, SUnSET labelling experiments show that global protein synthesis is unchanged in pcm¹⁴ mutant wing imaginal discs compared to wild-type. However, it cannot be ruled out that there may be reductions in protein translation for specific mRNAs. Another mechanism involves induction of cell death by overexpression of the apoptotic gene reaper using Beadex-Gal4, which has been demonstrated to result in upregulation of dilp8 transcripts by 13-fold in L3 wandering larvae. Previously published results show that pacman null mutations result in a post-transcriptional upregulation of reaper by 7.8-fold at the RNA level and an increase in apoptosis in the wing pouch (Waldron, 2015). The concomitant increase of dilp8 transcript by 6600-fold is consistent with the idea that, in wild-type imaginal discs, Pacman preferentially degrades both dilp8 and reaper transcripts to control both tissue growth and apoptosis (Jones, 2016).

The results detailed in this paper, together with our previous findings, show that Pacman regulates dilp8, Nplp2 and reaper transcripts at the post-transcriptional level. These transcripts range from 452-901 nt in length suggesting that short transcripts encoding small peptides are particularly susceptible to degradation by Pacman. The mechanisms by which these transcripts are specifically regulated by Pacman or its homologues in natural tissues are not yet clear. However, work in tissue culture cells suggest that mRNAs may be targeted to Pacman/XRN1 by specific RNA binding proteins and/or miRNAs. In rat pancreatic cells, it is known that polypyrimidine tract-binding protein binds to a pyrimidine-rich region of the insulin 3' UTR which contributes to the marked stability of insulin mRNA. Also, in rat cardiomyocytes, miR-1 targets the 3' UTR of IGF-1 reducing its expression. Therefore there are precedents for RNA-binding proteins/miRNAs regulating the expression of this family of proteins. Alternatively, mRNAs may be selectively sequestered in protein complexes which prevent access to ribonucleases and then be released for degradation via an unknown signal. The identification of a number of biologically relevant mRNAs regulated by Pacman presents an opportunity for understanding the mechanisms underlying the specificity of Pacman for certain mRNAs in a natural context (Jones, 2016).

A novel role for the 3'-5' exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth

This study has shown that the exoribonuclease Dis3L2 is required for regulation of proliferation in the wing imaginal discs in Drosophila. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3'-5' direction. Knockdown of Dis3L2 results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq, a small set of mRNAs was identified that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, two were identified that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). A compensatory effect between Dis3L2 and the 5'-3' exoribonuclease Pacman was identified, demonstrating that these two exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome (Towler, 2016).

Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper

Programmed cell death, or apoptosis, is a highly conserved cellular process that is crucial for tissue homeostasis under normal development as well as environmental stress. Misregulation of apoptosis is linked to many developmental defects and diseases such as tumour formation, autoimmune diseases and neurological disorders. This paper shows a novel role for the exoribonuclease Pacman/Xrn1 in regulating apoptosis. Using Drosophila wing imaginal discs as a model system, a null mutation in pacman was demonstrated to result in small imaginal discs as well as lethality during pupation. Mutant wing discs show an increase in the number of cells undergoing apoptosis, especially in the wing pouch area. Compensatory proliferation also occurs in these mutant discs, but this is insufficient to compensate for the concurrent increase in apoptosis. The phenotypic effects of the pacman null mutation are rescued by a deletion that removes one copy of each of the pro-apoptotic genes reaper, hid and grim, demonstrating that pacman acts through this pathway. The null pacman mutation also results in a significant increase in the expression of the pro-apoptotic mRNAs, hid and reaper, with this increase mostly occurring at the post-transcriptional level, suggesting that Pacman normally targets these mRNAs for degradation. These results uncover a novel function for the conserved exoribonuclease Pacman and suggest that this exoribonuclease is important in the regulation of apoptosis in other organisms (Waldron, 2015).

Apoptosis is a key process in developmental pathways and also in cancer. This study has generated a null mutation in pacman (pcm¹⁴ ) and used this to show that Pacman can control apoptosis in wing imaginal discs by regulating levels of hid and reaper mRNAs. Use of the Df(3L)H99 deletion, which removes one copy of the hid, grim and reaper genes, largely rescues the effect of the pcm¹⁴ mutation on growth of the wing imaginal discs and on developmental timing. However, the Df(3L)H99 deletion (Df(3L)H99/+) does not rescue the lethality of the pcm¹⁴ mutation. This suggests that there may be other targets of Pacman that are misregulated in pcm¹⁴ larvae or pupae (Waldron, 2015).

Mutant wing discs are proportionately reduced in size, even though the majority of apoptosis occurs in the wing pouch. The data also show that Pacman is expressed over the entire disc and pcm¹⁴ /pcm¹⁴ mutant clones are smaller than their wild-type twin spots throughout the disc. It is possible that apoptosis is occurring throughout the disc in earlier stages of development but is restricted to the wing pouch during L3. The data is consistent with other studies reporting that cells within the wing pouch are particularly sensitive to apoptosis, perhaps due to expression of particular apoptotic regulators in that region. The co-ordinate growth of the wing disc, even though apoptosis is occurring in a particular region of the disc, is likely to be due to long range signalling via morphogens which control overall patterning and growth of the wing disc. For example, Decapentaplegic (Dpp), a bone morphogenetic protein (BMP) functions as a long range morphogen to control patterning and growth. Furthermore, the Aegerter-Wilmsen model which explains how growth is constant throughout the disc suggests that growth of the peripheral cells within the disc is caused by stretching of the cells as a result of growth at the centre of the disc (see Aegerter-Wilmsen, 2012). Therefore, reduced growth at the centre of the disc, caused by apoptosis specifically in the pouch, is likely to cause reduced growth of the whole disc (Waldron, 2015).

The results also show that the pcm¹⁴ mutation induces cell proliferation as well as apoptosis. Apoptosis-induced compensatory proliferation is known to occur to maintain tissue homeostasis so that damaged tissues can be replaced allowing the organ to maintain its normal size. In Drosophila, this occurs via the initiator caspase Dronc which induces compensatory proliferation as well as apoptosis. Since Dronc is activated by Hid and Reaper, the increase in hid and reaper mRNA in pcm¹⁴ cells is consistent with increased activity of Dronc. Nevertheless, the 51%-54% increase in cell division in the pcm¹⁴ wing imaginal discs is insufficient to compensate for the concurrent increase in apoptosis because the wing discs fail to develop and differentiate, leading to death of the pupa. This failure of the wing discs to regenerate could be explained by there being prolonged apoptosis in the pcm¹⁴ wing imaginal discs, whereas other experiments have induced a pulse of apoptosis, allowing time for the wing disc to recover (Waldron, 2015).

The above results are consistent with reaper and hid being translated from the upregulated reaper and hid transcripts in pcm¹⁴ mutants. This would imply that these transcripts are both capped and polyadenylated. Biochemical analyses have shown that the less structured C-terminal domain of Pacman/Xrn1 includes short sections of conserved amino acids which bind co-factors such as the decapping protein Dcp1. Dcp1 associates with the decapping enzyme Dcp2, therefore coupling decapping to 5'-3' degradation. In pcm¹⁴ cells where no Pacman is present, decapping would therefore be expected to be impaired, which is consistent with our data. The alternative and/or additional hypothesis is that reaper and hid are being translated in a cap independent manner. Indeed the 5' UTRs of these genes have been shown to contain functional Internal Ribosome Entry Sites (IRES) and are still able to undergo translation in cells in which cap dependent translation is impeded (Waldron, 2015).

The above molecular mechanisms also are consistent with the 'dominant negative' effect seen when the nuclease-dead version of Pacman is expressed in a pcm¹⁴ mutant background. In Drosophila tissue culture cells, over-expression of catalytically inactive Pacman inhibited both decapping and degradation of a reporter RNA leading to an accumulation of capped fragments (Braun, 2012). Therefore the dominant negative effect could result from the sequestration of the Decapping protein Dcp1 together with lack of exonuclease activity. Expressing a 'nuclease dead' Pacman in pcm¹⁴ cells would not rescue any exoribonuclease activity but could impair decapping further. The results therefore support the model (Jones, 2012) that Pacman/Xrn1 normally assembles a complex of 5'-3' degradation factors including Dcp1 to provide a multicomponent complex which decaps and then degrades specific RNAs in a 5'-3' direction (Waldron, 2015).

The data, using natural tissue rather than immortalised tissue culture cells, supports the idea that there is a network of RNA-protein interactions contributing to apoptosis and proliferation. This idea is supported by work on the deadenylases Ccr4a and Ccr4b which can affect cell survival in MCF7 human breast cancer cells. Further, the RNA-binding protein HuR (homologue of Elav in Drosophila) has recently been shown to be cleaved in HeLa cells during caspase-mediated apoptosis with the two cleavage fragments binding to and stabilising caspase 9 mRNA, thus promoting apoptosis. The current data showing that the exoribonuclease Pacman is also involved in the control of apoptosis suggests a key role for the 5'-3' degradation pathway in the regulation of apoptosis (Waldron, 2015).

What are the mechanisms by which Pacman might be affecting the levels of mature hid and reaper mRNA? The simplest hypothesis is that Pacman is normally targeted to hid and reaper mRNA, resulting in degradation of these mRNAs. This targeting could be accomplished by specific RNA binding proteins and/or miRNAs binding to the 3' UTRs of hid and reaper mRNAs and directing them to the 5'-3' degradation machinery. The 3' untranslated regions of hid and reaper contain many predicted and validated miRNA binding sites for miRNAs. For example, the miRNA bantam has been shown to bind to the 3' UTR of hid mRNA, thus regulating its expression. In addition, miR-2 is known to bind to the 3' UTR of reaper, repressing its translation and directing it to P-body-like structures. A possible model to explain the results is that reaper and hid mRNAs are normally unstable because they are directed to the 5'-3' degradation complex by miRNAs binding to their 3' UTRs. In wild-type cells, these RNAs are rapidly decapped by decapping enzymes associated with Pacman and then degraded in a 5'-3' direction. In the Pacman mutant, these mRNAs are not efficiently degraded because of the absence of Pacman. It is also possible that reaper and hid are particularly affected by loss of Pacman because the presence of IRES sequences within their 5' UTRs means that these RNAs can be translated even if they are decapped. In a pacman mutant, these decapped RNAs may still be translated to produce Reaper and Hid protein. The exact mechanisms whereby Pacman regulates these mRNAs will require further research (Waldron, 2015).

The 5'-3' exoribonuclease Pacman (Xrn1) regulates expression of the heat shock protein Hsp67Bc and the microRNA miR-277-3p in Drosophila wing imaginal discs

Pacman/Xrn1 is a highly conserved exoribonuclease known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via miRNAs. Although Pacman has been well studied in Drosophila tissue culture cells, the biologically relevant cellular pathways controlled by Pacman in natural tissues are unknown. This study shows that a hypomorphic mutation in pacman (pcm) results in smaller wing imaginal discs. These tissues, found in the larva, are known to grow and differentiate to form wing and thorax structures in the adult fly. Using microarray analysis, followed by quantitative RT-PCR, it was shown that eight mRNAs were increased in level by>2-fold in the pcm⁵ mutant wing discs compared with the control. The levels of pre-mRNAs were tested for five of these mRNAs; four did not increase in the pcm mutant, showing that they are regulated at the post-transcriptional level and, therefore, could be directly affected by Pacman. These transcripts include one that encodes the heat shock protein Hsp67Bc, which is upregulated 11.9-fold at the post-transcriptional level and 2.3-fold at the protein level. One miRNA, miR-277-3p, is 5.6-fold downregulated at the post-transcriptional level in mutant discs, suggesting that Pacman affects its processing in this tissue. Together, these data show that a relatively small number of mRNAs and miRNAs substantially change in abundance in pacman mutant wing imaginal discs. Since Hsp67Bc is known to regulate autophagy and protein synthesis, it is possible that Pacman may control the growth of wing imaginal discs by regulating these processes (Jonmes, 2013).

A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5' exonucleolytic degradation

The removal of the mRNA 5' cap structure by the decapping enzyme DCP2 leads to rapid 5'-->3' mRNA degradation by XRN1, suggesting that the two processes are coordinated, but the coupling mechanism is unknown. DCP2 associates with the decapping activators EDC4 and DCP1. This study shows that XRN1 directly interacts with EDC4 and DCP1 in human and Drosophila melanogaster cells, respectively. In D. melanogaster cells, this interaction is mediated by the DCP1 EVH1 domain and a DCP1-binding motif (DBM) in the XRN1 C-terminal region. The NMR structure of the DCP1 EVH1 domain bound to the DBM reveals that the peptide docks at a conserved aromatic cleft, which is used by EVH1 domains to recognize proline-rich ligands. These findings reveal a role for XRN1 in decapping and provide a molecular basis for the coupling of decapping to 5'-->3' mRNA degradation (Braun, 2012).

The 5'-3' exoribonuclease Pacman is required for normal male fertility and is dynamically localized in cytoplasmic particles in Drosophila testis cells

The exoribonuclease Xrn1 is widely recognised as a key component in the 5'-3' RNA degradation pathway. This enzyme is highly conserved between yeast and humans and is known to be involved in RNA interference and degradation of microRNAs as well as RNA turnover. In yeast and human tissue culture cells, Xrn1 has been shown to be a component of P-bodies (processing bodies), dynamic cytoplasmic granules where RNA degradation can take place. This paper shows that Pacman, the Drosophila homologue of Xrn1, is localized in cytoplasmic particles in Drosophila testis cells. These particles are present in both the mitotically dividing spermatogonia derived from primordial stem cells and in the transcriptionally active spermatocytes. Pacman is co-localized with the decapping activator dDcp1 and the helicase Me31B (a Dhh1 homologue) in these particles, although this co-localization is not completely overlapping, suggesting that there are different compartments within these granules. Particles containing Pacman respond to stress and depletion of 5'-3' decay factors in the same way as yeast P-bodies, and therefore are likely to be sites of mRNA degradation or storage. Pacman is shown to be required for normal Drosophila spermatogenesis, suggesting that control of mRNA stability is crucial in the testis differentiation pathway (Zabolotskaya, 2008).

The 5'-3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered

Ribonucleases have been well studied in yeast and bacteria, but their biological significance to developmental processes in multicellular organisms is not well understood. However, there is increasing evidence that specific timed transcript degradation is critical for regulation of many cellular processes, including translational repression, nonsense-mediated decay and RNA interference. The Drosophila gene pacman is highly homologous to the major yeast exoribonuclease XRN1 and is the only known cytoplasmic 5'-3' exoribonuclease in eukaryotes. To determine the effects of this exoribonuclease in development a number of mutations in pacman was constructed by P-element excision, and the resulting phenotypes were characterized. Mutations in pacman resulted in flies with a number of specific phenotypes, such as low viability, dull wings, crooked legs, failure of correct dorsal/thorax closure and defects in wound healing. The epithelial sheet movement involved in dorsal/thorax closure is a conserved morphogenetic process which is similar to that of hind-brain closure in vertebrates and wound healing in humans. As the JNK (c-Jun N-terminal kinase) signalling pathway is known to be involved in dorsal/thorax closure and wound healing, this study tested whether pacman affects JNK signalling. The experiments demonstrate that pacman genetically interacts with puckered, a phosphatase that negatively regulates the JNK signalling pathway. These results reveal that the 5'-3' exoribonuclease pacman is required for a critical aspect of epithelial sheet sealing in Drosophila. Since these mutations result in specific phenotypes, the data suggest that the exoribonuclease Pacman targets a specific subset of mRNAs involved in this process. One of these targets could be a member of the JNK signalling pathway, although it is possible that a parallel pathway may instead be affected. The exoribonuclease Pacman is highly conserved in all eukaryotes, therefore it is likely that it is involved in similar morphological processes, such as wound healing in human cells (Grima, 2008).

A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing: Regulation of silencing by Ago1

In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1 (Pacman). These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins, which are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional, processes P-body or essential pathway components were depleted from Drosophila cells and the effects of these depletions were analyzed on the expression of reporter constructs, allowing specific monitoring of NMD, RNAi, or miRNA function. The RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. This analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila (Rehwinkel, 2005).

In eukaryotic cells, bulk messenger RNA (mRNA) is degraded via two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Following this first step, mRNAs can be degraded from their 3' ends by the exosome, a multimeric complex of 3' to 5' exonucleases. Alternatively, after deadenylation, the cap structure is removed by the DCP1:DCP2 decapping complex, and the mRNA is degraded by the major cytoplasmic 5' to 3' exonuclease XRN1 (Rehwinkel, 2005).

Proteins required for 5' to 3' mRNA degradation (e.g., DCP1, DCP2, and XRN1) colocalize in specialized cytoplasmic bodies or mRNA decay foci, also known as mRNA processing bodies (P-bodies) or GW-bodies, because of the accumulation of the RNA binding protein GW182 in these bodies. Additional components of P-bodies in yeast and/or human cells include the deadenylase Ccr4, the cap binding protein eIF4E and its binding partner eIF4E-transporter (eIF4E-T), auxiliary decay factors such as the LSm1-7 complex, Pat1p/Mtr1p, and the putative RNA helicase Dhh1/rck/p54. Among these, human GW182, eIF4E-T, and Dhh1 are required for P-body formation, while the decapping enzymes and XRN1 are dispensable. In addition, mRNA decay intermediates, microRNA (miRNA) targets, and miRNAs have been localized to P-bodies, suggesting that these bodies are sites where translationally silenced mRNAs are stored before undergoing decay (Rehwinkel, 2005 and references therein).

Recently, proteins involved in other post-transcriptional processes have been localized to P-bodies in human cells. These include the proteins SMG5, SMG7, and UPF1 involved in the nonsense-mediated mRNA decay (NMD) pathway and the Argonaute (AGO) proteins that play essential roles in RNA silencing. Moreover, XRN1 is recruited by both the NMD and the RNA interference (RNAi) machineries to degrade targeted mRNAs, suggesting a possible link between NMD, RNAi, and P-bodies. NMD is an mRNA quality control (or surveillance) mechanism that degrades aberrant mRNAs having premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. Core components of the NMD machinery include the proteins UPF1, UPF2, and UPF3, which form a complex whose function in NMD is conserved. The activity of UPF1 is regulated in multicellular organisms by additional proteins (i.e., SMG1, SMG5, SMG6, and SMG7) that are also required for NMD in all organisms in which orthologs have been characterized (Rehwinkel, 2005 and references therein).

In yeast and human cells, a major decay pathway for NMD substrates involves decapping and 5' to 3' degradation by XRN1. Although degradation of nonsense transcripts in Drosophila is initiated by endonucleolytic cleavage near the PTC, the resulting 3' decay intermediate is also degraded by XRN1. A molecular link between the NMD machinery and the decay enzymes localized in P-bodies is provided by SMG7 in human cells. Indeed, when overexpressed, human SMG7 localizes in P-bodies and recruits both UPF1 and SMG5 to these bodies, suggesting that NMD factors may reside at least transiently in P-bodies. RNA silencing pathways are evolutionarily conserved mechanisms that elicit decay or translational repression of mRNAs selected on the basis of complementarity with small interfering RNAs (siRNAs) or miRNAs, respectively. siRNAs are fully complementary to their targets and elicit mRNA degradation via the RNAi pathway. Animal miRNAs are only partially complementary to their targets and do not generally elicit decay, but repress translation instead (Rehwinkel, 2005 and references therein).

To perform their function, the siRNAs and miRNAs associate with the AGO proteins to form multimeric RNA-induced silencing complexes (RISC). Drosophila AGO1 mediates miRNA function, while AGO2 catalyzes the endonucleoytic cleavage of siRNA targets within the region complementary to the siRNA. Following this initial cleavage, the resulting 5' mRNA fragment is degraded by the exosome, while the 3' fragment is degraded by XRN1. The localization of AGO proteins in P-bodies in human cells provides a possible link between these bodies and silencing pathways (Rehwinkel, 2005 and references therein).

The NMD, the siRNA, and the miRNA pathways are therefore interlinked by the use of common decay enzymes and/or the coexistence of components of these pathways in P-bodies, suggesting a possible interdependence between these post-transcriptional mechanisms. Evidence for a link between NMD and RNAi has been reported in Caenorhabditis elegans where UPF1, SMG5, and SMG6 are required for persistence of RNAi, though not to initiate silencing. In contrast, UPF2, UPF3, and SMG1, which are also essential for NMD, are not required to maintain silencing, suggesting that UPF1, SMG5, and SMG6 may have evolved specialized functions in RNAi (Rehwinkel, 2005 and references therein).

This study investigates the interplay between NMD, RNAi, and the miRNA pathway using the Drosophila Schneider cell line 2 (S2 cells) expressing reporters allowing the monitoring of NMD, RNAi, or miRNA function. To this end, factors involved in NMD (UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6), RNAi (AGO2), or the miRNA pathway (AGO1) were depleted and the effect on the expression of the reporters analyzed. These proteins showed a high degree of functional specificity. To determine the role of P-body components in these pathways the DCP1:DCP2 decapping complex, the decapping coactivators LSm1 and LSm3, the 5' to 3' exonuclease XRN1, GW182, and the Drosophila protein CG32016, which shares limited sequence homology with human eIF4E-T, were depleted. The results uncovered a crucial role for GW182 and the DCP1:DCP2 decapping complex in the miRNA pathway (Rehwinkel, 2005).

Components of the NMD, RNAi, and miRNA pathways exhibit functional specificity in Drosophila To investigate a potential role of components of RNA silencing pathways or of P-body components in NMD, use was made of cell lines expressing wild-type or PTC-containing reporter constructs in which the coding regions of the bacterial chloramphenicol acetyl transferase (CAT) or the Drosophila alcohol dehydrogenase (adh) genes were placed downstream of inducible or constitutive promoters. The PTCs were inserted at codon 72 and 83 of the CAT and adh open reading frames, respectively. P-body components and proteins involved in NMD, RNAi, or the miRNA pathway were depleted by treating the cells with double-stranded RNAs (dsRNAs) specific for the different factors. A dsRNA that targets green fluorescent protein (GFP) served as a control. The steady-state levels of the wild-type and PTC-containing mRNAs were analyzed by Northern blot and normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32) (Rehwinkel, 2005).

Relative to the expression levels of the wild-type mRNAs, the levels of the corresponding PTC-containing transcripts are reduced because these transcripts are rapidly degraded via the NMD pathway. Depletion of UPF1 inhibits NMD, so the levels of the PTC-containing mRNAs are restored. Depletion of AGO1 or AGO2, both singly and in combination, does not interfere with the NMD pathway, although these depletions do inhibit siRNA- or miRNA-mediated gene silencing. The levels of the CAT wild-type transcript were not affected by the depletions. Similar results were obtained with the NMD reporter based on the adh gene. Together, these results indicate that inhibition of RNAi or of the miRNA pathway does not interfere with NMD. XRN1 is the only P-body component known to be required for degradation of decay intermediates arising from mRNAs undergoing NMD in Drosophila. Nevertheless, in cells depleted of XRN1 the NMD pathway is not inhibited, and only the 3' decay intermediate generated by endonucleolytic cleavage of the mRNA accumulates (Rehwinkel, 2005).

In contrast to XRN1, none of the P-body components tested, including GW182 and the DCP1:DCP2 decapping complex, affected NMD or the accumulation of the 3' decay intermediate. The lack of a significant effect of the depletion of the DCP1:DCP2 complex was confirmed using the adh reporter. The decapping enzymes are certainly involved in NMD in yeast and human cells because the major decay pathway for NMD substrates is initiated by decapping in these organisms (for review, see Conti, 2005). Thus, it is possible that the requirement for P-body components and/or P-body integrity in NMD varies across species (Rehwinkel, 2005).

Two different approaches were used to investigate the RNAi pathway. In one approach, a cell line constitutively expressing the wild-type Drosophila adh gene was treated with a dsRNA complementary to a central region of ~300 nucleotides (nt) of adh mRNA (adh dsRNA). This dsRNA elicits decay of the adh mRNA via the RNAi pathway. Cells were treated with dsRNAs targeting various factors in the presence or absence of adh dsRNA. The steady-state levels of the adh mRNA were analyzed by Northern blot and normalized to those of the rp49 mRNA. In cells treated with GFP dsRNA, the normalized levels of the adh transcript were reduced to 4% after addition of adh dsRNA, relative to the levels detected in the absence of adh dsRNA. In cells depleted of AGO2, a sixfold increase of adh mRNA levels was observed despite the presence of adh dsRNA. In contrast, when AGO1 was depleted, adh dsRNA could still trigger a reduction of adh mRNA levels, though a slight increase in transcript levels was observed. Similarly, depletion of UPF1 did not prevent silencing of adh expression by adh dsRNA. These results indicate that UPF1 is not required for RNAi in Drosophila. Additional NMD components (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6) have been identified, but no SMG7 ortholog has been identified in Drosophila. No significant change was observed in the efficacy of RNAi under the conditions in which NMD was inhibited (Rehwinkel, 2005).

Similarly to the results reported for the NMD pathway, depletion of XRN1 leads to the accumulation of the 3' decay intermediate generated by endonucleolytic cleavage by RISC, while depletion of the DCP1:DCP2 decapping complex does not prevent RNAi or the degradation the 3' decay intermediate. In contrast, depletion of GW182 leads to a modest increase in the adh mRNA level in the presence of adh dsRNA, suggesting that this protein could influence the efficiency of RNAi (Rehwinkel, 2005).

In a second approach, RNAi was triggered by an siRNA instead of a long dsRNA, to uncouple RISC activity from processing of dsRNAs. To this end, S2 cells were transiently transfected with a plasmid expressing firefly luciferase (F-Luc) and an siRNA targeting the luciferase coding sequence (F-Luc siRNA) or a control siRNA. A plasmid encoding Renilla luciferase (RLuc) was included to normalize for transfection efficiencies. Cotransfection of the F-Luc reporter with the F-Luc siRNA led to a 50-fold inhibition of firefly luciferase activity relative to the activity measured when the control siRNA was cotransfected, indicating that F-Luc siRNA effectively silences firefly luciferase expression (Rehwinkel, 2005).

The results obtained with the luciferase reporter correlate well with those obtained with adh mRNA, in spite of differences between the methods used to detect changes in reporter levels (RNA levels vs. protein levels), and the nature of the RNA trigger (long dsRNA vs. siRNA). Indeed, depletion of AGO2 impaired silencing of firefly luciferase expression by the F-Luc siRNA, leading to an eightfold increase in firefly luciferase activity relative to the activity of the Renilla control. Depletion of AGO1 led to a twofold increase of firefly luciferase activity (Rehwinkel, 2005).

The observation that depletion of AGO2, but not AGO1, significantly inhibits RNAi is in agreement with previous reports showing that only AGO2-containing RISC is able to catalyze mRNA cleavage triggered by siRNAs. The results together with these observations indicate that Drosophila AGO1 and AGO2 are not redundant (Rehwinkel, 2005).

Depletion of GW182 or the DCP1:DCP2 complex led to a 1.5- to twofold increase of the firefly luciferase activity, although RNAi was not abolished. These results together with those obtained with the adh reporter suggest that GW182 and the DCP1:DCP2 complex are not absolutely required for RNAi but may modulate siRNA function (Rehwinkel, 2005).

Finally, depletion of core NMD components does not inhibit the silencing of firefly luciferase expression by F-Luc siRNA. The results are consistent with results from C. elegans showing that NMD per se is not required for the establishment of silencing (Rehwinkel, 2005).

To investigate the miRNA pathway firefly luciferase reporters were generated in which the coding region of firefly luciferase is flanked by the 3' UTRs of the Drosophila genes CG10011 or Vha68-1. These genes were identified as miRNA targets in a genome-wide analysis of mRNAs regulated by AGO1. The 3' UTR of CG10011 mRNA contains two binding sites for miR-12, while the 3' UTR of Vha68-1 has two binding sites for miR-9b. Expression of the firefly luciferase construct fused to the 3' UTR of CG10011 (F-Luc-CG10011) was strongly reduced by cotransfection of a plasmid expressing the primary (pri) miR-12 transcript, but not pri-miR-9. Conversely, expression of the firefly luciferase reporter fused to the 3' UTR of Vha68-1 (FLuc-Vha68-1) was inhibited by cotransfection of pri-miR-9b, but not of primiR-12 (Rehwinkel, 2005).

Silencing of luciferase expression by the cognate miRNAs was prevented in cells depleted of AGO1. Indeed, despite the presence of the transfected miRNAs, in cells depleted of AGO1 an 11-fold and a 16-fold increase of firefly luciferase expression was observed from the FLuc- CG10011 and F-Luc-Vha68-1 reporters, respectively. Notably, the firefly luciferase activity measured in AGO1-depleted cells in the presence of the transfected miRNAs was at least twofold higher than the activity measured in control cells in the absence of exogenously added miRNAs. Since endogenous miR-9b and miR-12 are expressed in S2 cells, these results suggest that depletion of AGO1 also suppresses silencing mediated by the endogenous miRNAs. Depletion of AGO2 does not suppress the effect of coexpressing the reporters with the cognate miRNAs. These results provide additional evidence supporting the conclusion that the siRNA and miRNA pathways are not interdependent (Rehwinkel, 2005).

miRNA-mediated silencing of firefly luciferase expression was not affected by depletion of UPF1 or by the additional NMD factors (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6). Thus, the individual NMD factors and NMD per se are not required for miRNA function. Unexpectedly, although the efficiency of NMD and RNAi was unaffected or only modestly affected in cells depleted of GW182 or the DCP1:DCP2 complex, miRNA-mediated silencing of firefly luciferase expression was effectively relieved in these cells. In the presence of cognate miRNAs, depletion of GW182 resulted in a sixfold increase of firefly luciferase expression. Therefore, despite the presence of transfected miRNAs, firefly luciferase activity in GW182-depleted cells was similar to that measured in controls cells in the absence of transfected miRNAs. Codepletion of DCP1 and DCP2 led to a fourfold increase of firefly luciferase expression. Finally, depletion of CG32016 resulted in a twofold increase of firefy luciferase activity, but only for the F-Luc-Vha68-1 reporter, suggesting that this effect may not be significant (Rehwinkel, 2005).

To investigate whether depletion of GW182 affects RISC activity directly, as opposed to interfering with miRNA processing, use was made of a tethering assay. This assay involves the expression of a lN-fusion of AGO1 that binds with high affinity to five BoxB sites (5-BoxB) in the 3' UTR of a firefly luciferase reporter mRNA. When AGO1 is tethered to this reporter transcript, luciferase expression is inhibited relative to the activity measured in cells expressing the lN-peptide alone. The inhibition was partially relieved in cells depleted of GW182 but not of AGO2. It is concluded that GW182 and the decapping DCP1: DCP2 complex play a critical role in the effector step of the miRNA pathway. These results are in agreement with the observation that Argonaute proteins localize to P-bodies and interact with DCP1 and DCP2 independently of RNA or of P-body integrity (Rehwinkel, 2005).

Thus, despite convergence in P-bodies, NMD, RNAi, and the miRNA pathway are not interdependent in Drosophila. This conclusion is based on the observation that the inhibition of one pathway by depleting key effectors may slightly interfere with, but does not significantly inhibit, the functioning of the other pathways. The lack of interdependence between RNAi and the miRNA pathway is further supported by the observation that knockouts of AGO1 or AGO2 in Drosophila have different phenotypes. Nevertheless, cross-talk between the RNAi and the miRNA pathways may still occur at the initiation step, since Dicer-1 plays a role in RISC assembly (Rehwinkel, 2005).

Biochemical and genetic approaches in several organisms have led to the identification of essential components of the miRNA pathway. These include AGO1 and the enzymes required for miRNA processing, such as Drosha and Dicer-1 and their respective cofactors, Pasha and Loqs. However, the mechanisms by which miRNAs inhibit protein expression without affecting mRNA levels are not completely understood. Recent evidence suggests that translation initiation is inhibited and that the targeted mRNAs are stored in P-bodies, where they are maintained in a silenced state either by associating with proteins that prevent translation or possibly by removal of the cap structure. This study identified the P-body components GW182 and the DCP1:DCP2 decapping complex as proteins required for the miRNA pathway. The precise molecular mechanism by which these proteins participate in this pathway remains to be established. These proteins may have an indirect role in the miRNA pathway by affecting P-body integrity. Alternatively, these proteins may play a direct role in this pathway by escorting miRNA targets to P-bodies or facilitating mRNP remodeling steps required for the silencing of these targets. Consistent with a direct role for the DCP1:DCP2 decapping complex, and thus for the cap structure, in miRNA function is the observation that mRNAs translated via a cap-independent mechanism are not subject to miRNA-mediated silencing. In conclusion, the results uncover an important role for the P-body components, GW182 and the DCP1:DCP2 complex, in miRNA-mediated gene silencing (Rehwinkel, 2005).

Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. This study has identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (Pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring Pacman exoribonuclease activity in yeast, it was demonstrate that Pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. These experiments provide the first example of an exoribonuclease which is differentially expressed throughout development (Till, 1998).

Search PubMed for articles about Drosophila Pacman

Aegerter-Wilmsen, T., Heimlicher, M. B., Smith, A. C., de Reuille, P. B., Smith, R. S., Aegerter, C. M. and Basler, K. (2012). Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size. Development 139(17): 3221-3231. PubMed ID: 22833127

Braun, J. E., Truffault, V., Boland, A., Huntzinger, E., Chang, C. T., Haas, G., Weichenrieder, O., Coles, M. and Izaurralde, E. (2012). A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5' exonucleolytic degradation. Nat Struct Mol Biol 19(12): 1324-1331. PubMed ID: 23142987

Grima, D. P., Sullivan, M., Zabolotskaya, M. V., Browne, C., Seago, J., Wan, K. C., Okada, Y. and Newbury, S. F. (2008). The 5'-3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered. Biol Cell 100(12): 687-701. PubMed ID: 18547166

Jindra, M., Gaziova, I., Uhlirova, M., Okabe, M., Hiromi, Y. and Hirose, S. (2004). Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila. EMBO J 23(17): 3538-3547. PubMed ID: 15306851

Jones, C. I., Zabolotskaya, M. V. and Newbury, S. F. (2012). The 5' --> 3' exoribonuclease XRN1/Pacman and its functions in cellular processes and development. Wiley Interdiscip Rev RNA 3(4): 455-468. PubMed ID: 22383165

Jones, C. I., Grima, D. P., Waldron, J. A., Jones, S., Parker, H. N. and Newbury, S. F. (2013). The 5'-3' exoribonuclease Pacman (Xrn1) regulates expression of the heat shock protein Hsp67Bc and the microRNA miR-277-3p in Drosophila wing imaginal discs. RNA Biol 10(8): 1345-1355. PubMed ID: 23792537

Jones, C. I., Pashler, A. L., Towler, B. P., Robinson, S. R. and Newbury, S. F. (2016). RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1. Nucleic Acids Res 44(1): 267-280. PubMed ID: 26656493

Li, F. Q., Ueda, H. and Hirose, S. (1994). Mediators of activation of fushi tarazu gene transcription by BmFTZ-F1. Mol Cell Biol 14(5): 3013-3021. PubMed ID: 8164657

Lim, A. K., Tao, L. and Kai, T. (2009). piRNAs mediate posttranscriptional retroelement silencing and localization to pi-bodies in the Drosophila germline. J Cell Biol 186(3): 333-342. PubMed ID: 19651888

Liu, Q. X., Jindra, M., Ueda, H., Hiromi, Y. and Hirose, S. (2003). Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems. Development 130(4): 719-728. PubMed ID: 12506002

Nishioka, K., Wang, X. F., Miyazaki, H., Soejima, H. and Hirose, S. (2018). Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman. Development 145(5). PubMed ID: 29523653

Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. and Izaurralde, E. (2005). A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11(11): 1640-7. PubMed ID: 16177138

Takemaru, K., Li, F. Q., Ueda, H. and Hirose, S. (1997). Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein. Proc Natl Acad Sci U S A 94(14): 7251-7256. PubMed ID: 9207077

Takemaru, K., Harashima, S., Ueda, H. and Hirose, S. (1998). Yeast coactivator MBF1 mediates GCN4-dependent transcriptional activation. Mol Cell Biol 18(9): 4971-4976. PubMed ID: 9710580

Till, D. D., Linz, B., Seago, J. E., Elgar, S. J., Marujo, P. E., Elias, M. L., Arraiano, C. M., McClellan, J. A., McCarthy, J. E. and Newbury, S. F. (1998). Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster. Mech Dev 79(1-2): 51-55. PubMed ID: 10349620

Towler, B. P., Jones, C. I., Harper, K. L., Waldron, J. A. and Newbury, S. F. (2016). A novel role for the 3'-5' exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth. RNA Biol [Epub ahead of print]. PubMed ID: 27630034

Waldron, J. A., Jones, C. I., Towler, B. P., Pashler, A. L., Grima, D. P., Hebbes, S., Crossman, S. H., Zabolotskaya, M. V. and Newbury, S. F. (2015). Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper. Biol Open 4(5): 649-660. PubMed ID: 25836675

Zabolotskaya, M. V., Grima, D. P., Lin, M. D., Chou, T. B. and Newbury, S. F. (2008). The 5'-3' exoribonuclease Pacman is required for normal male fertility and is dynamically localized in cytoplasmic particles in Drosophila testis cells. Biochem J 416(3): 327-335. PubMed ID: 18652574

date revised: 15 March 2020

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