Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 5

Figure 5. Pths increases assembled ribosome levels to stimulate the translation of an mRNA subset with short 5' UTRs

A Sucrose density gradient fractionation allowed purification of ribosome subunits and polysomes. Polysomal or total cellular mRNA fractions were isolated following dsRNA treatment and RNA sequencing libraries were prepared.

B Sedimentation analysis showing the relative abundance of 40S, 60S, and 80S ribosomes in cells treated with a gfp-dsRNA (control) or porthos (pths)-dsRNA. Profiles were aligned by the 40S ribosome peak's position. Black: control, Red: pths-KD. n=3 independent biological replicates.

C Scatter plot of Translational efficiency (TE) from pths-dsRNA S2R+ vs control gfp-dsRNA cells. Red (down-regulated, DR) and green (up-regulated, UP) dots represent genes with log2TE changes that meet the 2 standard deviation cutoff.

D Bioinformatic analysis shows that mRNAs whose TE is increased by Porthos have significantly shorter 5'UTRs than non-targets (p=1.69e-35). Box and whisker plot of log2(Mean 5'UTR length) of each genotype. Middle line of box represents median, upper and lower bounds of box represents 1st and 3rd quartiles of data. Whiskers represent 1.5*IQR (inner quartile range), points outside of 1.5*IQR are plotted explicitly. The corresponding table shows median/mean 5'UTR lengths in base pairs (bp) for all mRNAs expressed in S2 cells (non-targets) and for the subset whose TE is enhanced by Porthos (Porthos targets). Targets were defined using n=3 biological replicates of polysome-seq. 5'UTR length comparison is between n=8451 non-target genes and n=204 Porthos targets.

E-F Confocal images of early Stage 12 embryos from the controls, from lines expressing nlacZ and RNAis against atos or pths in macrophages (mac>) as a second control, and lines expressing RNAis targeting atos or pths along with individual components of the dTORC1 pathway in macrophages, including Nrpl2, Iml1, and TSC1. Germband edge: dotted white line.

G-H Quantification of the number of germband macrophages in embryos from the controls and upon RNAi knockdown of the genes in E-F. Expression of different RNAis against Nrpl2, Iml1, and TSC1 in atos or pths-depleted macrophages rescues macrophage invasion into the germband while expression of lacZ does not. In (G) control (n=21 embryos) vs. atos RNAi (n=30) p<0.0001; control vs. atos RNAi rescued with Nrpl2 RNAi (n=32) p=0.019, with Iml1 RNAi (n=24) p=0.03, and with TSC1 RNAi (n=15) p=0.04. In (H) control (n=29 embryos) vs pths RNAi (n=26) p<0.0001; control vs. pths RNAi rescued with Nrpl2 RNAi (n=27) p=0.01, with Iml1 RNAi (n=17) p=0.02, and with TSC1 RNAi (n=18) p=0.02.

I Western blot of protein extracts from control and pths KD S2R+ probed with p-4EBP1 antibody. Tubulin serves as a loading control and quantification normalized to loading control. N=3 biological replicates for both control and pths KD, p=0.69.

Data information: Mean±SEM, ns=p>0.05, *p<0.05, ****p<0.0001. Welch's t-test (D), unpaired t-test (I), one-way ANOVA with Tukey (G-H). Scale bar: 30µm (E-F). See Source Data 1 and 2 for Figure 5.