Burnett, S. B. | bioRxiv Science

bioRxiv2025

Genetic background influences the effects of RNAi-by-feeding for DNA methyltransferases in Daphnia pulex.

DNA methyltransferases (DNMTs) are well-characterized epigenetic enzymes responsible for transferring methyl groups to and from DNA. Three main DNMT orthologues differ in function and methylation capability. They each are evolutionarily conserved across diverse taxa, but few studies investigate them jointly. We did so in Daphnia, freshwater microcrustaceans used extensively in research on genetic diversity, phenotypic plasticity, and maternal effects. Most Daphnia reproduce asexually through cyclic parthenogenesis, making the Daphnia system an ideal choice for studying epigenetic phenomena. Advances in gene expression control techniques, including RNA interference (RNAi), have increased the versatility and power of the Daphnia system. RNAi is a post-transcriptional gene silencing mechanism that operates through sequence-specific cleavage of endogenous messenger RNA (mRNA) transcripts. Here, we used an RNAi bacterial feeding regime to target the three DNA methyltransferase genes in two clones of Daphnia pulex. We observed significant genotypic differences in response to the RNAi feeding regime, namely, the mortality of one clone. In the other, DNMT expression significantly increased in five of the six experimental treatments, with the highest level observed in animals treated with the GFP double-stranded RNA bacterial vector control. However, DNMT expression was reduced in all three DNMT RNAi treatments relative to the GFP control. Furthermore, we found strong cross-reactivity, where targeting one DNMT resulted in the reduction of the other two. This response may be associated with known immune pathways involving signal transduction that can be stimulated by viral and bacterial signals, or it may result from previously unknown aspects of DNMT biology.

genetics↗

bioRxiv2024

A frameshift mutation in the murine Prkra gene causes dystonia and exhibits abnormal cerebellar development and reduced eIF2α phosphorylation.

Mutations in Prkra gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of the subunit of the eukaryotic translation initiation factor 2 (eIF2). Mice homozygous for a naturally arisen, recessively inherited frameshift mutation, Prkralear-5J exhibit progressive dystonia. In the present study, we investigate the biochemical and developmental consequences of the Prkralear-5J mutation. Our results indicate that the truncated PACT/RAX protein retains its ability to interact with PKR, however, it inhibits PKR activation. Furthermore, mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons. Additionally, reduced eIF2 phosphorylation is noted in the cerebellums and Purkinje neurons of the homozygous Prkralear-5J mice. These results indicate that PACT/RAX mediated regulation of PKR activity and eIF2 phosphorylation plays a role in cerebellar development and contributes to the dystonia phenotype resulting from this mutation. Summary StatementThis study shows, for the first time, a role of reduced eIF2 phosphorylation in DYT-PRKRA and the cerebellum development in a mouse model.

molecular biology↗