A Functional Screen Reveals an Extensive Layer of Transcriptional and Splicing Control Underlying RAS/MAPK Signaling in Drosophila

Ashton‐Beaucage, Dariel; Udell, Christian M.; Gendron, Patrick; Sahmi, Malha; Lefrançois, Martin; Baril, Caroline; Guenier, Anne-Sophie; Duchaine, Jean; Lamarre, Daniel; Lemieux, Sébastien; Therrien, Marc

doi:10.1371/journal.pbio.1001809

Cited by 60 publications

(85 citation statements)

References 130 publications

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“…Biochemical studies indicate that Nito, like its human ortholog, copurify with the precatalytic spliceosome (complex B) (34). In addition, nito, as well as many other splicing factors, was identified in an RNAi screen for RAS/MAPK signaling components (35). Consistent with these findings, we find that nito is required for the alternative splicing of the master sex-determination gene Sxl.…”

Section: Discussionsupporting

confidence: 81%

spenito is required for sex determination in Drosophila melanogaster

Dong

Perrimon

2015

Proc. Natl. Acad. Sci. U.S.A.

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Sex-lethal (Sxl) encodes the master regulator of the sex determination pathway in Drosophila and acts by controlling sex identity in both soma and germ line. In females Sxl maintains its own expression by controlling the alternative splicing of its own mRNA. Here, we identify a novel sex determination gene, spenito (nito) that encodes a SPEN family protein. Loss of nito activity results in stem cell tumors in the female germ line as well as female-to-male somatic transformations. We show that Nito is a ubiquitous nuclear protein that controls the alternative splicing of the Sxl mRNA by interacting with Sxl protein and pre-mRNA, suggesting that it is directly involved in Sxl auto-regulation. Given that SPEN family proteins are frequently mutated in cancers, our results suggest that these factors might be implicated in tumorigenesis through splicing regulation.germ-line stem cell | sex determination | alternative splicing S ex determination in Drosophila is under the control of the master regulatory gene Sex-lethal (Sxl) (1). Sxl acts downstream of the X-chromosome counting mechanism and encodes a female-specific RNA binding protein. Once activated, Sxl maintains its own expression by regulating the alternative splicing of its pre-mRNA. Sxl controls female fate by controlling somatic and germ-line sex identity as well as dosage compensation (2). In female somatic cells, Sxl controls the alternative splicing of transformer (tra), which together with transformer2 (tra2) controls the alternative splicing of doublesex (dsx) and fruitless (fru). dsx and fru in turn encode sex-specific transcription factors that control male versus female morphology, physiology, and behavior (3, 4). In addition, Sxl represses the male-specific dosage compensation system by regulating male-specific lethal 2 (msl-2) both at the level of alternative splicing and translational control (5

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Section: Discussionsupporting

confidence: 81%

spenito is required for sex determination in Drosophila melanogaster

Dong

Perrimon

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, we identified several splicing factors, including Prp19 and Prp8, in a genome-wide RNAi screen in S2 cells for modulators of Ras-induced MAPK activation (Ashton-Beaucage et al 2014). Incidentally, a single mutant allele of Prp8 was also recovered in the CCT screen (reported in Ashton-Beaucage et al 2014). Characterization of their implication in the pathway revealed that they specifically regulate MAPK protein levels by controlling the alternative splicing of selected introns of the mapk pre-mRNAs.…”

Section: Modifiers Of Dominant-negative Cnkmentioning

confidence: 99%

Apical Accumulation of the Sevenless Receptor Tyrosine Kinase During Drosophila Eye Development Is Promoted by the Small GTPase Rap1

et al. 2014

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The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/ MAPK signaling is well understood, far less is known regarding its interplay with other corequired signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity.

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“…(B) Epistasis analysis in Drosophila S2 cells employing constitutively active forms of RAS, RAF, and MEK to induce MAPK activation [8]. Phosphorylated MAPK was measured by quantitative microscopy and normalized to a GFP dsRNA control [7].…”

Section: Introductionmentioning

confidence: 99%

The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in Drosophila

et al. 2016

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RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate Drosophila MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity.

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A Functional Screen Reveals an Extensive Layer of Transcriptional and Splicing Control Underlying RAS/MAPK Signaling in Drosophila

Abstract: A global RNAi screening approach in Drosophila cells identifies a large group of transcription and splicing factors that modulate RAS/MAPK signaling by altering the expression of MAPK.

Cited by 60 publications

References 130 publications

spenito is required for sex determination in Drosophila melanogaster

spenito is required for sex determination in Drosophila melanogaster

Apical Accumulation of the Sevenless Receptor Tyrosine Kinase During Drosophila Eye Development Is Promoted by the Small GTPase Rap1

The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in Drosophila

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