Smg binds to mRNAs encoding mitochondrial proteins and is involved in mRNA down-regulation due to PABPN1-17ala expression.
A) Quantification of mRNA levels in control and PABPN1-17ala-expressing thoraxes in the presence or absence of heterozygous twin or smg mutations at day 6, using RT-qPCR. mRNA levels were normalized to Cpr100A mRNA. Means of two biological replicates quantified three times. Error bars represent standard deviation. * p-value <0. 05, ** p-value <0.01, *** p-value <0.001, ns: not significant, using the Student’s t-Test. B) Confocal images of immunostaining of indirect flight muscles from wild type and smg mutant (smgPL00423/Df(3L)scf-R6) adult flies with anti-Smg (green). DNA was visualized with DAPI (blue). Smg protein levels were strongly reduced in smgPL00423/Df(3L)scf-R6 muscles compared to wild-type muscles. Scale bars: 5 μm. C) Western blots of protein extracts from wild-type and smg mutant thoraxes revealed with guinea pig (gp) and rabbit (rb) anti-Smg, showing the presence of Smg and its lower level in smg mutant. α-Tubulin was used as a loading control. D) Smg immunoprecipitations (IP) in UASp-CCR4-HA/Mhc-Gal4 adult thoraxes, either in the presence or the absence of RNase A. Mock IP was with rabbit IgG. Input is the protein extract prior to immunoprecipitation. Western blots revealed with anti-Smg, anti-HA and anti-PABP2, showing CCR4-HA co-precipitation and the lack of PABP2 co-precipitation. E) Quantification of mRNA enrichment in Smg IP using RT-qPCR. The ratio of mRNA/control mRNA was set to 1 in the mock IP (black line). Normalization was with sop mRNA. RpL32 mRNA is a negative control, which is not enriched in Smg IP. Means are from two independent IP quantified three times. Error bars represent standard error to the mean. F) Means of SRE scores of mRNAs down-regulated in PABPN1-17ala-expressing muscles and annotated with the term "mitochondrion" compared to those of control mRNAs, in Drosophila (left panel) and mouse (right panel). Twenty times 98 Drosophila and 10 times 407 mouse control genes were used. Error bars represent standard deviation.
