ADAD1 and ADAD2, testis-specific adenosine deaminase domain-containing proteins, are required for male fertility

Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.


Special considerations regarding testicular RNA editing
Multiple factors have been identified as regulators of RNA editing with the best studied being the AD-domain proteins themselves. This report and previous related work (19) shows that while germ cell RNA editing is catalyzed by a known RNA editing enzyme, other AD-domain containing proteins do not appear to regulate germ cell RNA editing. These findings suggest other mechanisms of control may be at play. While cis elements within RNA editing targets have dramatic impacts on RNA editing efficiency by giving rise to specific RNA secondary structures (28), it seems unlikely that cis elements within the germ cell transcriptome alone explain the relative lack of RNA editing. This is especially true given the complexity of the male germ cell transcriptome exceeds even that of the brain (20, 24), a site of extremely high RNA editing levels. However, RNA editing has been shown to be quite sensitive to a cell's complement of RNA binding proteins (RBPs) (29) and male germ cells express an incredibly wide range of RBPs (30), in part due to their tight reliance on post-transcriptional processes for normal differentiation (21). RBPs, which drive an extremely diverse range of biological process, act on the transcriptome and as such can change the availability, structure, localization, and stability of RNA editing targets in a celltype or tissue-dependent manner. Which, if any, germ cell-specific RBPs interact with the RNA editing pathway remains an open question, however efforts leveraging RNA-sequencing data from large-scale projects like ENCODE may provide valuable insight.
In addition to individual RBPs, it has been demonstrated that entire RNA processing pathways intersect and impact RNA editing. In particular, splicing has been shown to have a robust influence on RNA editing for a large number of targets (29). The interaction of these two pathways is reasonable given RNA editing is known to occur co-transcriptionally (7) and is often the result of exonic and intronic cis elements interacting to form the double stranded RNA template used by ADAR enzymes (30).
Consistent with this, a recent report demonstrated that global reduction of splicing leads to significant increases in editing efficiency (31) while mutation of specific alternative splicing factors altered editing of more specific targets, with decreased splicing generally being associated with increased editing. These findings are buoyed by the observation that a large number of alternative splicing proteins have been identified as RBP regulators of RNA editing (27). The interaction of splicing and RNA editing may be especially important in the context of the male germ cell where alternative splicing is particularly high (20), which mechanistically may serve to globally repress RNA editing. To facilitate the depth of alternative splicing observed, the testis expresses a wide range of splicing factors, some or many of which may influence total or site-specific RNA editing. As germ cells develop, they leverage different suites of alternative splicing programs (32) to facilitate cell-specific alternative splicing patterns across germ cell development. This cell-specific splicing regulation will require careful dissection to identify potential splicing regulators of germ cell RNA editing.
Phenotypic differences between Adad1 tm1Reb relative to Adad1 em2 may be driven by multiple mechanisms While it is possible the difference in phenotype between Adad1 tm1Reb and Adad1 em2 may be due to differences between wildtype ADAD1 and the abnormal proteins detected in the Adad1 tm1Reb testes, it seems most likely the reduced phenotypic severity in Adad1 tm1Reb relative to Adad1 em2 is due to a reduction, but not total loss, of functional ADAD1 protein in Adad1 tm1Reb . Further, this outcome may be exacerbated by a number of other mechanisms. As with all CRISPR generated alleles, it is feasible carrier mutations in the background of the CRISPR allele are leading to a more severe phenotype.
However, this is extremely unlikely as extensive analyses confirmed no carrier mutations at other probable CRISPR targets within the genome. A much more likely mechanisms is that the two alleles behave differently as a function of their slightly different genetic context. Although both mutant models were derived from the same strain, due to breeding the two alleles were analyzed on slightly different genetic backgrounds. This hypothesis is supported by previous observations (18) showing that when moved to a different genetic background, the Adad1 tm1Reb fertility phenotype changed dramatically. These suggest ADAD1 function may be reliant on genetic modifiers and open the possibility of defining ADAD1's molecular function via genome-scale modifier screens.