Identification and Spatiotemporal Expression of Adenosine Deaminases Acting on RNA (ADAR) during Earthworm Regeneration: Its Possible Implication in Muscle Redifferentiation.

Abstract

Simple SummaryAmong the animal species capable of regenerating missing body parts, a species of earthworm, Perionyx excavatus, has the most powerful regeneration capacity, which can completely and regenerate an amputated head and tail. Earthworm regeneration is a form of epimorphosis, a simple mode of development in adults that occurs around the sites of damage rather than throughout the body. In order to achieve this process, the earthworm must have molecular tools via which a variety of cell and tissue types can be precisely recovered from the pluripotent (or possibly totipotent) blastemal cells. Adenosine to inosine (A-to-I) RNA editing catalyzed by adenosine deaminases acting on RNA (ADAR) can generate substantial transcriptome and proteome variability and provide an ideal tool for cell and tissue re-specification. To understand the role of ADAR during earthworm regeneration, the molecular characteristics of an ADAR gene identified from P. excavatus (Pex-ADAR) were analyzed, and its spatial and temporal expression patterns were observed during regeneration. Domain analysis showed that Pex-ADAR is a member of the ADAR1 class. Its expression level primarily increases when and where muscle redifferentiation is actively taking place, suggesting that the RNA-editing enzyme Pex-ADAR is involved in muscle redifferentiation.Adenosine deaminases acting on RNA (ADAR) catalyze the hydrolytic deamination of adenosine (A) to produce inosine (I) in double-stranded RNA substrates. A-to-I RNA editing has increasingly broad physiological significance in development, carcinogenesis, and environmental adaptation. Perionyx excavatus is an earthworm with potent regenerative potential; it can regenerate the head and tail and is an advantageous model system to investigate the molecular mechanisms of regeneration. During RNA sequencing analysis of P. excavatus regenerates, we identified an ADAR homolog (Pex-ADAR), which led us to examine its spatial and temporal expression to comprehend how Pex-ADAR is linked to regeneration. At first, in domain analysis, we discovered that Pex-ADAR only has one double-stranded RNA-binding domain (dsRBD) and a deaminase domain without a Z-DNA-binding domain (ZBD). In addition, a comparison of the core deaminase domains of Pex-ADAR with those of other ADAR family members indicated that Pex-ADAR comprises the conserved three active-site motifs and a glutamate residue for catalytic activity. Pex-ADAR also shares 11 conserved residues, a characteristic of ADAR1, supporting that Pex-ADAR is a member of ADAR1 class. Its temporal expression was remarkably low in the early stages of regeneration before suddenly increasing at 10 days post amputation (dpa) when diverse cell types and tissues were being regenerated. In situ hybridization of Pex-ADAR messenger RNA (mRNA) indicated that the main expression was observed in regenerating muscle layers and related connective tissues. Taken together, the present results demonstrate that an RNA-editing enzyme, Pex-ADAR, is implicated in muscle redifferentiation during earthworm regeneration.