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.

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.

Author Summary

The RAS/MAPK pathway is a cornerstone of the cell proliferation signaling apparatus. It has a notable involvement in cancer as mutations in the components of the pathway are associated with aberrant proliferation. Previous work has focused predominantly on post-translational regulation of RAS/MAPK signaling such that a large and intricate network of factors is now known to act on core pathway components. However, regulation at the pre-translational level has not been examined nearly as extensively and is comparatively poorly understood. In this study, we used an unbiased and global screening approach to survey the Drosophila genome—using Drosophila cultured cells—for novel regulators of this pathway. Surprisingly, a majority of our hits were associated to either transcription or mRNA splicing. We used a series of secondary screening assays to determine which part of the RAS/MAPK pathway these candidates target. We found that these factors were not equally distributed along the pathway, but rather converged predominantly on mapk mRNA expression and processing. Our findings raise the intriguing possibility that regulation of mapk transcript production is a key step for a diverse set of regulatory inputs, and may play an important part in RAS/MAPK signaling dynamics.