Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m <sup>6</sup> A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m <sup>6</sup> A alters fly behavior, albeit the underlying molecular mechanism and the role of m <sup>6</sup> A during nervous system development have remained elusive. Here we find that impairment of the m <sup>6</sup> A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m <sup>6</sup> A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m <sup>6</sup> A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m <sup>6</sup> A pathway controls development of the nervous system and modulates Fmr1 target transcript selection

High-throughput micro-characterization of RNA-protein interactions

12 p.-2 fig.-2 tabMany cellular processes depend on and are regulated by nucleic acid-protein interactions. In particular, RNA-binding proteins (RBPs) are involved in transcription, translation, modulating RNA polymerase activity, and stabilizing protein-RNA complexes. Furthermore, RBPs participate in the development of pathologies such as cancer and viral infections, and their dysfunction leads to mutations and the aberrant expression of noncoding RNAs. Therefore, the study of RNA-protein interactions represents a central issue for biology and biomedicine. While many valuable insights have been obtained from electrophoretic mobility shift assays (EMSA) and immunoprecipitation (IP), these standard methods suffer from two main limitations: insufficient sensitivity to capture low concentration RBP-RNA complexes in vitro and identification of interactions in vivo. In recent years, high-throughput (HTP) platforms have emerged that combine methodological improvements over conventional techniques with more sensitive detection systems, thereby catalyzing the simultaneous probing and analysis of a vast amount of RBP-RNA interactions by cellular proteomics and interactomics approaches. In this chapter, we summarize a selection of state-of-the-art in vitro, in vivo, and computational HTP platforms for the discovery and characterization of RNA-protein interactions. We also reflect on the wealth of information obtained by the structural analysis of RBPs and their RNA-binding domains as a valuable resource for the rational design and implementation of new RNA-binding discovery platforms.MCV has received funding from the Spanish Ministerio de Economía y Competitividad (CTQ2015-66206-C2-2-R and SAF2015-72961-EXP) and the Regional Government of Madrid (S2017/BMD-3673).Peer reviewe