Precursor miRNAs are locally processed to regulate growth cone steering

During the development of the nervous system, axons grow and generate a complex network of interconnected neurons. To establish these connections, the tip of the growing axon, the growth cone, is guided by chemotropic cues en route to its target with exquisite precision. Axons must sometimes navigate a significant distance before reaching their final destination. As an alternative to energy-expensive protein transport from distant cell bodies, seminal studies have revealed that growth cones rely on local mRNA translation to generate certain proteins acutely on demand. These cue-induced newly synthesized proteins contribute to fuel growth cone steering. Several groups reported the presence of Dicer at growth cones, and I observe the presence of endogenous Dicer in RGC axons of FLAG-HA2-Dicer transgenic mice. These observations raise the intriguing possibility that not only proteins but also miRNAs could be produced locally in this compartment. In my work, I have therefore explored whether miRNA biogenesis occurs locally within growth cone and if this is important for growth cone steering, using Xenopus laevis retinal ganglion cell (RGC) axons as a model. Specific precursor microRNAs (pre-miRNAs) are detected in pure Xenopus RGC axonal preparations by miRNA-seq and PCR, and endogenous pre-miR-181a-1 is actively tracked to distal axons by hitchhiking on vesicles. Upon exposure to Sema3A, but not Slit-2, pre-miR-181a-1/a-2 are processed within axons by Dicer into newly generated miRNAs (NGmiRNAs). In contrast, pre-miR-182 remains unprocessed upon Sema3A exposure, highlighting a mechanism that is not only cue-, but also pre-miRNA molecule specific. Inhibiting NGmiRNAs in axons abolishes growth cone responsiveness to cues ex vivo. miRNAs are thus locally produced and these newly generated miRNAs mediate cue-induced growth cone steering. To deepen mechanistic insights, I assess whether newly generated miRNAs silence the translation of specific mRNAs in response to cues using FRAP analysis with a Venus reporter. I ...