The lncRNA male-specific abdominal plays a critical role in Drosophila accessory gland development and male fertility
Fig 6
miR iab-8 loss-of-function causes defects in the LTR.
Two different fertility fecundity assays were used to assess the LTR of mutants lacking the iab-8 miRNA: (A. and B.) egg-laying over the 10 days post-mating and (C. and D.) receptivity after four days post-mating. The ΔmiR-iab-8 mutation was tested over two different iab-6cocu alleles to overcome possible effects of genetic background iab-6cocuD1(D1) or iab-6cocuD5 (D5). For the 10-day egg laying assay, in A., the D1 curve (in blue, n = 15) refers to iab-6cocuD1homozygous males, the D1Resc curve (in red, n = 18) refers to iab-6cocuD1/ iab-5,6rescue males, where iab-5,6rescue is a chromosome made with the same InSiRT platform [70] used to make the iab-6cocu mutations but with wild-type sequence added in its place. The mirResc curve (in green, n = 22) refers to ΔmiR-iab-8/ iab-5,6rescue males and the mirD1 curve (in purple, n = 16) refers to iab-6cocuD1/ ΔmiR-iab-8 males. In B., the D5 curve (in blue, n = 18) refers to iab-6cocuD5 homozygous males, the D5Resc curve (in red, n = 22) refers to iab-6cocuD5/ iab-5,6rescue males, the mirResc curve (in green, same as in A., n = 22) refers to ΔmiR-iab-8/ iab-5,6rescue males and the mirD5 curve (in purple, n = 21) refers to iab-6cocuD5/ ΔmiR-iab-8 males. In both cases there is a significant drop in egg laying in mates of either iab-6cocu mutant or in mates of males transheterozygous for either iab-6cocu mutant and the ΔmiR-iab-8 chromosome (rmANOVA p>0.001 relative to controls). The receptivity assay was performed on the same genotypes. In C. and D., sample sizes are as follows, D1 (n = 19), D1 Resc (n = 18), mir Resc (n = 20) mirD1 (n = 20), D5 (n = 19), D5 Resc (n = 20), mir Resc (same as in C., n = 20) and mirD5 (n = 20). In both cases, there is a significant increase in remating (p < .05) by mates of either iab-6cocu mutants or in mates of males transheterozygous for either iab-6cocu mutant and the ΔmiR-iab-8 chromosome (* indicates p < .007 vs D1 res, p≤.02 mir Res and p < .001 D1 relative to controls, ** indicates p < .001 relative to controls, as accessed by the Wilcoxin Ranked Sums Test). E. Shows extracts from single AGs run on Western blots and probed with antibodies against CG1656 or CG1652. Below these blots are images of the same blots stripped and reprobed with a loading control antibody against the main cell protein, Acp36DE. The genotype of the flies from which the AGs were dissected are indicated above each lane and the antibodies used indicated on the left. F. Shows western blots of extracts from wild type or mutant AGs probed with antibodies against CG1656. The extracts on the left were treated with PGNase F, while those on the right were not. Genotypes of the flies used for the extracts are indicated above each lane. Below the blot is an image of the same blot stripped and reprobed with a loading control antibody against the main cell protein, Acp36DE.