InteractiveFly: GeneBrief

viriato: Biological Overview | References

The nucleolus is a subnuclear factory, the activity of which is required beyond ribosome biogenesis for the regulation of cell growth, death and proliferation. In both Drosophila and mammalian cells, the activity of the nucleolus is regulated by the proto-oncogene Myc. Myc induces the transcription of genes required for ribosome biogenesis and the synthesis of rRNA by RNA polymerase I, a nucleolar event that is rate limiting for cell growth. This study shows that the fruit fly Nol12 homologue Viriato is a key determinant of nucleolar architecture that is required for tissue growth and cell survival during Drosophila development. It is further shown that viriato expression is controlled by Drosophila Myc (dMyc), and that the ability of dMyc to stimulate nucleolar and cellular growth depends on viriato expression. Therefore, viriato acts downstream of dMyc to ensure a coordinated nucleolar response to dMyc-induced growth and, thereby, normal organ development (Marinho, 2011).

Tissue and organ development require a precise coordination of cellular growth, proliferation, differentiation and apoptosis. At the core of the cell, and crucial for its growth, the nucleolus is the subnuclear compartment where ribosome biogenesis takes place (Boisvert, 2007). Cell mass accumulation is required for proliferation, implying that the regulation of nucleolar function plays an important role in the control of proliferation rates. In the nucleolus, RNA polymerase I (Pol I) synthesizes pre-rRNAs, which are processed, modified and assembled in 40S and 60S pre-ribosomal particles that are then exported to the cytoplasm. Besides its role as the ribosome factory, the nucleolus is now also considered to be a multifunctional regulatory compartment involved in RNA processing events, sensing of cell stress, and cell cycle and apoptosis regulation (Boisvert, 2007; Marinho, 2011 and references therein).

A key step in ribosome biogenesis is pre-rRNA gene transcription by Pol I, a process that in human cells is known to be stimulated by the binding of c-MYC to rRNA promoters in the nucleolus (Arabi, 2005; Grandori, 2005). Further, Drosophila Myc (dMyc; also known as Diminutive), which is known to control the cell cycle and apoptosis, has been shown to be necessary and sufficient for the transcription of genes encoding Pol I transcription machinery factors, such as Tif-IA and RpI135 (the largest Pol I subunit), genes encoding pre-rRNA processing and modifying factors, such as Nop60B and Fibrillarin, as well as a large set of ribosomal genes (Grewal, 2005; Pierce, 2008). The ability of dMyc to induce a coordinated nucleolar hypertrophy and to stimulate pre-rRNA transcription and ribosome biogenesis in general are required for dMyc-stimulated growth during Drosophila development (Grewal, 2005). This study identifies viriato (vito) as a dMyc target gene that coordinates nucleolar and growth responses downstream of dMyc (Marinho, 2011).

This study shows that dMyc controls vito mRNA levels to regulate nucleolar architecture and that vito is required for dMyc to reach its full potential as a potent cell growth inducer. Furthermore, the knockdown of vito expression also correlated with an increase in p53-independent, caspase-mediated apoptotic cell death, suggesting a potential novel link between structural and functional changes in the nucleolus and activation of the pro-apoptotic rpr/grim/hid complex (Marinho, 2011).

During development, dMyc plays a crucial role in translating intracellular and extracellular cues to regulate the pace of cell growth and proliferation. One of the main mechanisms for dMyc-stimulated growth appears to be the transcriptional control of nucleolar ribosome biogenesis genes (Grewal, 2005; Hulf, 2005; Teleman, 2008; Demontis, 2009). Cells of the salivary glands are polyploid secretory cells with very active biosynthetic pathways. In these cells, increasing or reducing Vito levels results in changes in the nucleolar localisation patterns of the pre-rRNA methyltransferase Fibrillarin and in alterations in nucleolar structure. The fact that vito does not appear necessary for the expression of dMyc targets implicated in ribosomal biogenesis suggests that part of the control that dMyc exerts on the nucleolus is mediated independently of its regulation of vito expression. In addition, several results support the hypothesis that the Myc-Nol12 regulatory relationship is evolutionarily conserved. Genome-wide chromatin immunoprecipitation analysis has shown that c-MYC binds the NOL12 promoter in both a human transformed B-cell line and in mouse stem cells. Non-canonical E-box motifs (CACATG) have been identified in the putative proximal promoter regions of both vito and human NOL12 (Marinho, 2011).

In addition to its function in cell growth downstream of dMyc, vito plays a role in supporting the proliferation and survival of diploid cells. dMyc mutants are smaller than the wild type, and dMyc mutant cells grow poorly in the context of wild-type tissue. Therefore, vito is a rate-limiting factor for tissue growth that links dMyc with nucleolar architecture. The mechanisms enacting this link might prove relevant for the regulation of Myc function in tumourigenesis (Marinho, 2011).

The nucleolar protein Viriato/Nol12 is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development

Drosophila Decapentaplegic (Dpp), a member of the BMP2/4 class of the TGF-betas, is required for organ growth, patterning and differentiation. However, much remains to be understood about the mechanisms acting downstream of these multiple roles. This issue was investigated during the development of the Drosophila eye. viriato (vito) has been identified as a dMyc-target gene encoding a nucleolar protein that is required for proper tissue growth in the developing eye. By carrying out a targeted in vivo double-RNAi screen to identify genes and pathways functioning with Vito during eye development, a strong genetic interaction was found between vito and members of the Dpp signaling pathway including the TGF-beta receptors tkv (type I), put (type II), and the co-Smad medea (med). Analyzing the expression of the Dpp receptor Tkv and the activation pattern of the pathway's transducer, p-Mad, vito was found to be required for a correct signal transduction in Dpp-receiving cells. Overall, this study validated the use of double RNAi to find specific genetic interactions and, in particular, a link between the Dpp pathway and Vito, a nucleolar component, was uncovered. vito would act genetically downstream of Dpp, playing an important role in maintaining a sufficient level of Dpp activity for the promotion of eye disc growth and regulation of photoreceptor differentiation in eye development (Marinho, 2013).

Different genetic relationships are uncovered by the detection of aggravating synthetic interactions. A pair of genes could act in parallel pathways converging on the same biological process ('between-pathway' interaction), or could either act at the same level or different levels of one pathway ('within-pathway' interaction). Ultimately, it is also possible that each gene may act in unrelated processes revealing an indirect interaction, even though the breakdown of the system occurs when both genes are compromised. Within this conceptual framework, this study reports the first in vivo double-RNAi screen to study genetic interactions during Drosophila development, providing evidence for the usefulness of tissue-targeted RNAi screens for the detection of aggravating synthetic genetic interactions. An in vivo double-RNAi was performed screen to uncover genes and pathways functioning with the nucleolar regulator Vito during eye development and 12 interactor genes were identified. Eleven out of the 12 Vito interactor genes identified have been described, or predicted, to be involved in the development of the nervous system. Furthermore, a significant interaction was detected between vito and the retinal determination genes ey, eya, and so. However these interactions are weaker (lower interaction scores) than the interactions between vito and Dpp signaling genes. Dpp and Eya (this latter partnering with So, the Six2 homolog) are both required downstream of Hh for retinogenesis. Both Dpp and Eya/So are then required for further differentiation of the retina and the repression of hth, a transcription factor that maintains the progenitor state. Dpp and Hh are also required redundantly to establish So expression. Thus, the interaction between vito and the retinal determination genes could potentially be a consequence of the significant modulation of Dpp signaling by vito. Interestingly, it was observed that vito interacts with members of the TGF-β signaling pathway, including the Dpp signaling pathway receptors tkv and put, but also with members of the activin signaling branch, such as the R-Smad smox/dSmad2 and the receptor type-I baboon. Recent knowledge about the cross-talk between the activin and Dpp branches is arising as it was shown that Smox (the activin dedicated R-Smad) has a role in wing disc growth that requires the function of Mad. Moreover, it was also reported that Baboon, the type-I activin receptor, is able to phosphorylate the Dpp branch dedicated Mad in a Smox-concentration dependent manner. These reports hint at the complex inter-regulation between both branches of the TGF-β signaling, which complicates the detailed analysis of the exact contribution of vito to the signaling activities of the two branches (Marinho, 2013).

Taken together, the data demonstrate that Vito acts downstream of Dpp, having a dual role during eye development: Vito cooperates with Dpp in growth stimulation during early stages of eye disc development and also in later stages during the process of eye disc patterning. Vito/Dpp interaction does not seem to be based on Vito's requirement for survival in the developing eye because no increase in the number of apoptotic cells was detected when Vito was depleted together with the Dpp receptor tkv. The vito-Dpp interaction specifically takes place in the context of eye development where vito is required for Myc-stimulated growth. To assess a potential interaction between the Dpp pathway and Myc in the developing eye, double-RNAi experiments were carried that revealed a synthetic interaction between dMyc and Med. However, the interaction observed after co-depleting vito and med is even stronger. These results point to a specific and direct interaction of vito with the Dpp signaling pathway that is not simply an indirect effect from the previously described dMyc-vito interaction (Marinho, 2013).

Overall, these data are consistent with a role of Vito in positively regulating Dpp signaling since depletion of Vito partially reverted Dpp overexpression phenotypes, and the phenotype of depleting vito in a Dpp weak RNAi background resembled a strong RNAi for a Dpp pathway component. Remarkably, overexpression of low levels of Vito could rescue the absence of differentiation caused by a strong reduction in Dpp activity by put RNAi. Moreover, vitoRNAi eye discs showed a delay in MF progression and an irregular activation of p-Mad within the furrow, which was accompanied with a reduction in Tkv levels. Whether Vito regulates Dpp signaling by direct modulation of Tkv levels, or whether this down-regulation of Tkv is an indirect effect due to a decreased signaling output from Dpp signaling remains an open question. In conclusion, the genetic data reveal that Vito, a nucleolar putative RNA 5'-3' exonuclease, modulates Dpp signaling during fly eye development. Extensively known for its role in ribosome biogenesis, recent studies suggest that the nucleolar sub-nuclear compartment is also linked to cell-cycle and developmental decisions. As an example, Nucleostemin is a nucleolar GTP-binding protein with both ribosomal and non-ribosomal roles, and was recently shown to maintain self-renewal of embryonic stem cells and to play a role in injured-induced liver regeneration. As a further example, differentiation of primary spermatocytes into mature spermatids was shown to require the nucleolar sequestration of Polycomb repression complex 1 factors. Provocatively, one of the reports by the ENCODE project that surveyed the transcriptome of nuclear subcompartments in the K562 cell line revealed that a small fraction of transcripts with distinct GO-enrichment is unique to the nucleolar compartment. Although the genetic data do not reveal the molecular mechanism underlying the Vito/Dpp interaction, it is interesting to note that alterations of Vito expression levels have profound effects on nucleolar architecture. In the face of the dynamic and potentially important role of the nucleolus in the control of gene expression, the interaction between Dpp and vito could result from altered expression or sub-nuclear localization of yet to be identified regulators of Dpp signaling when vito expression is knocked-down. Thus, further experiments are necessary to elucidate the mechanisms underlying the role of Vito in the modulation of Dpp functions in Drosophila eye development (Marinho, 2013).

Search PubMed for articles about Drosophila Viriato

Arabi, A., Wu, S., Ridderstrale, K., Bierhoff, H., Shiue, C., Fatyol, K., Fahlen, S., Hydbring, P., Soderberg, O., Grummt, I., Larsson, L. G. and Wright, A. P. (2005). c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat Cell Biol 7: 303-310. PubMed ID: 15723053

Boisvert, F. M., van Koningsbruggen, S., Navascues, J. and Lamond, A. I. (2007). The multifunctional nucleolus. Nat Rev Mol Cell Biol 8: 574-585. PubMed ID: 17519961

Demontis, F. and Perrimon, N. (2009). Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila. Development 136: 983-993. PubMed ID: 19211682

Grandori, C., Gomez-Roman, N., Felton-Edkins, Z. A., Ngouenet, C., Galloway, D. A., Eisenman, R. N. and White, R. J. (2005). c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nat Cell Biol 7: 311-318. PubMed ID: 15723054

Grewal, S. S., Li, L., Orian, A., Eisenman, R. N. and Edgar, B. A. (2005). Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development. Nat Cell Biol 7: 295-302. PubMed ID: 15723055

Hulf, T., Bellosta, P., Furrer, M., Steiger, D., Svensson, D., Barbour, A. and Gallant, P. (2005). Whole-genome analysis reveals a strong positional bias of conserved dMyc-dependent E-boxes. Mol Cell Biol 25: 3401-3410. PubMed ID: 15831447

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

Marinho, J., Martins, T., Neto, M., Casares, F. and Pereira, P. S. (2013). The nucleolar protein Viriato/Nol12 is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development. Dev Biol 377: 154-165. PubMed ID: 23416177

Pierce, S. B., Yost, C., Anderson, S. A., Flynn, E. M., Delrow, J. and Eisenman, R. N. (2008). Drosophila growth and development in the absence of dMyc and dMnt. Dev Biol 315: 303-316. PubMed ID: 18241851

Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab 7: 21-32. PubMed ID: 18177722

date revised: 25 July 2016

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