Visualisation of ribosomes in Drosophila axons using Ribo-BiFC

Abstract

8Rates of protein synthesis and the number of translating ribosomes vary greatly between 9 different cells in various cell states. The distribution of assembled, and potentially 10 translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular 11Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 12 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-subunit 13 junctions in the assembled 80S ribosome. Here we describe transgenes that express two 14 novel RP pairs tagged with Venus-based BiFC fragments that considerably increase the 15 sensitivity of this technique that we termed Ribo-BiFC. This improved method should 16 provide a convenient way of monitoring the local distribution of ribosomes in most 17Drosophila cells and we suggest that could be implemented in other organisms. We 18 visualized 80S ribosomes in larval photoreceptors and in other neurons. Assembled 19 ribosomes are most abundant in the various neuronal cell bodies, but they are also present 20 along the lengths of axons and are concentrated in growth cones of larval and pupal 21 photoreceptors. Surprisingly, there is relatively less puromycin incorporation in the distal 22 portion of axons in the optic stalk, suggesting that some of the ribosomes that have 23 started translation may not be engaged in elongation in axons that are still growing. 24Running title: Ribosomes in axon growth cones 25

The cap epitranscriptome: Early directions to a complex life as mRNA

“…In the absence of cOMe in Drosophila , both the CBC and EJC are reduced at synapses indicating that cOMe provides a mark for certain mRNAs to be transported in an untranslated state to synapses for local translation. [ 14,71 ]…”

“…In the absence of cOMe in Drosophila, both the CBC and EJC are reduced at synapses indicating that cOMe provides a mark for certain mRNAs to be transported in an untranslated state to synapses for local translation. [14,71] Intriguingly, CMTr2 targets in Drosophila are enriched in transcripts also targeted by FMR1, the homologue of human FMRP which has been shown to regulate protein synthesis. [14,72] In fact, the translational repressive role of FMRP in arresting ribosomes on mRNAs is consistent with a role of cOMe in directing untranslated mRNAs to synapses.…”