Daphnia magna egg piRNA cluster expression profiles change as mothers age.

Abstract

PiRNAs prevent transposable elements wreaking havoc on the germline genome. Changes in piRNA expression over the lifetime of an individual may impact on ageing through continued suppression, or release, of transposable element expression. We identified piRNA producing clusters in the genome of Daphnia magna by a combination of bioinformatic methods, and then contrasted their expression between parthenogenetically produced eggs representing maternally-deposited germline piRNAs of young (having their 1st clutch) and old (having their 5th clutch) mothers. Results from eggs were compared to cluster expression in three generations of adults. As for other arthropods, D. magna encodes long uni-directionally transcribed non-coding RNAs which consist of transposable element fragments which account for most piRNAs expressed. Egg tissues showed extensive differences between clutches from young mothers and those from old mothers, with 578 and 686 piRNA clusters upregulated, respectively, although most log fold-change differences for significant clusters were modest. When considering only highly expressed clusters, there was a bias towards 1st clutch eggs at 41 upregulated versus eight clusters in the eggs from older mothers. F0 generation differences between young and old mothers were fewer than eggs, as 179 clusters were up-regulated in young versus 170 old mothers. This dropped to 31 versus 22 piRNA clusters when comparing adults in the F1 generation, and no differences were detected in the F3 generation. These patterns were similar to that observed for D. magna micro-RNA expression. Little overlap in differentially expressed clusters was found between adults containing mixed somatic and germline (ovary) tissues and germ-line representing eggs. A cluster encompassing a Tudor domain containing gene important in the piRNA pathway was upregulated in the eggs from old mothers. We hypothesise that regulation of this gene this could form part of a feedback loop that reduces piRNA pathway activity explaining the reduced number of highly-expressed clusters in eggs from old mothers. Author SummarySmall RNAs shorter than 200 nucleotides often function by targeting RNAs with complementary nucleotide sequences for destruction. A subset of small RNAs, the Piwi-interacting RNAs or piRNAs are best known for silencing sequences of DNA that can jump between locations in the genome which can compromise the integrity of genomes. This protects offspring from sterility and other undesirable effects, hence piRNAs have been termed guardians of the genome. PiRNAs have several characteristics, such as a characteristic length and origin in genomic graveyards of junk DNA, that can be used to distinguish them from other small RNAs. Here, we used a combination of computational approaches to identify piRNA-producing clusters in the genome of the water flea Daphnia magna. We then contrasted expression of these clusters between genetically identical adults of different ages, their eggs, adult offspring and great-granddaughters. Adults and their eggs were markedly different in cluster expression by age, which was mostly lost by adulthood in offspring and not seen at all in great-granddaughters. By taking an innovative approach that can be applied to similar datasets of diverse organisms we have shown that piRNA expression, and therefore, stability of the genome can change with age.