title: A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.
abstract: The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing.
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Dariel
authors.initials: D
authors.lastname: Ashton-Beaucage
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Udell
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Gendron
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Malha
authors.initials: M
authors.lastname: Sahmi
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Lefrançois
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Caroline
authors.initials: C
authors.lastname: Baril
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Guenier
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Duchaine
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada; Département de médecine, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Lamarre
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada; Département d'informatique et de recherche opérationnelle, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lemieux
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Montréal, Québec, Canada; Département de pathologie et de biologie cellulaire, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Marc
authors.initials: M
authors.lastname: Therrien
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D016000:Cluster Analysis; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D004843:Epistasis, Genetic; D005786:Gene Expression Regulation; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020935:MAP Kinase Signaling System; D034622:RNA Interference; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D020930:MAP Kinase Kinase Kinases; D018631:ras Proteins