Figure 6. Cauda-specific microRNAs, but not tRFs, rescue Caput preimplantation defects.
A) Schematic representation of microinjection experiment. As in Figure 5A, but two separate small RNA populations were gel-purified – 18–26 nt RNAs are enriched for microRNAs (but include additional molecular species including short 5’ and 3’ tRFs), while 26–40 nt RNAs are primarily comprised of tRNA fragments.
B) Purification of epididymosomal RNAs. Acrylamide electrophoresis of two replicate total RNA samples purified from cauda epididymosomes. Boxes show the boundaries used for gel-purification of microRNAs and tRFs.
C) mRNA abundance for individual 4-cell stage Cauda (n=23), Caput (n=23), Caput+microRNA (n=22), and Caput+tRF (n=21) embryos, for representative Caput-upregulated genes. In all cases, cauda-specific “microRNAs” returned mRNA abundance to Cauda levels, while tRFs had little to no effect on these target genes. See Supplemental Figure S6B for blastocyst-stage data. The clear difference between these matched RNA injections – both RNA samples should be free of contaminating proteins, and should include similar levels of any potential contaminants such as leftover acrylamide – demonstrates that the rescue activity is specific to 18–26 nt cauda epididymosomal small RNAs.
D) Scatterplot comparing Caput effects (x axis) and microRNA effects (y axis) on mRNA abundance in 4-cell stage embryos. See Supplemental Figure S6C for blastocyst stage dataset.
E–F) Histograms of microRNA and tRF effects on Caput-upregulated genes. In both cases, data are shown for mRNAs which exhibit a log2 fold change of at least 0.5 in Caput vs. Cauda embryos (p<0.01, uncorrected for multiple hypothesis testing to include a greater number of genes – results are even more dramatic for the smaller subset of genes significant after multiple hypothesis-correction). For both 4-cell stage (E) and blastocyst-stage (F) data, cauda-specific microRNAs cause downregulation of Caput-induced genes, while tRFs have little to no effect on these genes (KS p value comparing microRNA and tRF effects < 10−15 for both datasets).