The majority of A-to-I RNA editing is not required for mammalian homeostasis

Background Adenosine-to-inosine (A-to-I) RNA editing, mediated by ADAR1 and ADAR2, occurs at tens of thousands to millions of sites across mammalian transcriptomes. A-to-I editing can change the protein coding potential of a transcript and alter RNA splicing, miRNA biology, RNA secondary structure and formation of other RNA species. In vivo, the editing-dependent protein recoding of GRIA2 is the essential function of ADAR2, while ADAR1 editing prevents innate immune sensing of endogenous RNAs by MDA5 in both human and mouse. However, a significant proportion of A-to-I editing sites can be edited by both ADAR1 and ADAR2, particularly within the brain where both are highly expressed. The physiological function(s) of these shared sites, including those evolutionarily conserved, is largely unknown. Results To generate completely A-to-I editing-deficient mammals, we crossed the viable rescued ADAR1-editing-deficient animals (Adar1E861A/E861AIfih1−/−) with rescued ADAR2-deficient (Adarb1−/−Gria2R/R) animals. Unexpectedly, the global absence of editing was well tolerated. Adar1E861A/E861AIfih1−/−Adarb1−/−Gria2R/R were recovered at Mendelian ratios and age normally. Detailed transcriptome analysis demonstrated that editing was absent in the brains of the compound mutants and that ADAR1 and ADAR2 have similar editing site preferences and patterns. Conclusions We conclude that ADAR1 and ADAR2 are non-redundant and do not compensate for each other’s essential functions in vivo. Physiologically essential A-to-I editing comprises a small subset of the editome, and the majority of editing is dispensable for mammalian homeostasis. Moreover, in vivo biologically essential protein recoding mediated by A-to-I editing is an exception in mammals.

For more microscopic details, please refer to individual animals in the report below. Notes: (1) Accumulation of leukocytes and other cells are common nonneoplastic lesions in many tissues. The term "inflammation" is used when the cell (leukocyte) accumulations are part of an active inflammatory process (typified by concurrent features such as vascular changes, necrosis, fibrosis, and/or tissue disruption). In contrast, cell "infiltration" is used when the cell (e.g., lymphocyte) accumulations are present in tissue without other disruption or pathology.
(2) Focal inflammatory cell aggregates consisting of mononuclear, polymorphonuclear, and/or histiocytic cells are frequently observed in ageing mice (Maranport RR. 1999. Pathology of the Mouse.). These can be present as lymphoid aggregates found in various tissues including the renal pelvis, bladder, lungs, liver (Pettan-Brewer C and Treuting PM. 2011. Practical pathology of aging mice.) and salivary glands (Haines DC, Chattopadhyay S and Ward JM. 2001. Pathology of Aging B6;129 Mice).
(3) Reactive lymph nodes are defined as mild follicular hyperplasia, germinal centre formation and occasional sinus histiocytosis -a common finding in mice.
(4) Hyperplastic lymph node follicles are identified by an increase in number and size of follicles and conversion to secondary follicles. Hyperplasia of the paracortex is characterized by an increase in the cell density and, depending on the degree of hyperplasia, an increase in the paracortical area. (5) Mild extramedullary haematopoiesis (EMH) identified in the red pulp of all the spleens, a common finding in the mouse. EMH consists of erythroid precursors, myeloid precursors, megakaryocytes or all three. While some degree of extramedullary haematopoiesis is present in normal rodents, especially in mice, increased extramedullary haematopoiesis can result from haematotoxin insult, systemic anaemia, and infections elsewhere in the body. (Suttie AW. 2006. Histopathology of the spleen). (6) Hyperplasia of the stomach epithelium is defined as an increase in the number of cells in one or more epithelial strata (basal, spinous, or granulosum). Hyperkeratosis (increased thickness of the stratum corneum) is often seen when hyperplasia of the mucosa is present. Hyperplasia of the nonglandular stomach is often characterized as a uniform thickening of all layers of the epithelium without upward or downward projections. However, in more severe cases there are convolutions of the epithelium with downward projections of rete peg-like structures. Focal upward proliferation into the lumen mimicking papilloma may be present in more severe cases of focal hyperplasia and are described as papillary hyperplasia (Kooistra LH, Nyska A. 2014. Stomach, Forestomach, Epithelium -Hyperplasia. In: National Toxicology Program Nonneoplastic Lesion Atlas). (7) Thymic cysts in the rodent represent either a dilatation of thymic tubular structures or remnants of the thymopharyngeal duct. They are common findings in the involuted and/or atrophied thymus glands of rats and mice. Thymic cyst formation becomes more prominent with age and is associated with involution. (Hobbie K, Elmore SA and Kolenda-Roberts HM. 2015. Thymus -Cyst. In: National Toxicology Program Nonneoplastic Lesion Atlas). (8) Valvular myxomatous changes or degeneration can be an age-related spontaneous or chemicalinduced change. The lesion is characterized by focal or segmental thickening of the subendocardium in the valve leaflets and expansion of the spongiosa of the valve leaflet with extracellular fibromyxoid material composed predominantly of glycosaminoglycans. Occasionally, fibrin deposits or thrombi and collections of neutrophils or mononuclear cells are seen (Johnson CL, Nyska A. 2017. Heart, Valve, -Degeneration. In: National Toxicology Program Nonneoplastic Lesion Atlas). (9) Germ cell degeneration of the testes is a nonspecific term that generally includes a number of degenerative features, such as tubular vacuolation, partial depletion of germ cells, degenerating (multinucleated or apoptotic) germ cells, and disordered arrangement of the germ cell layers. Chemically induced germ cell degeneration can be multifocal in distribution, but it is most often a bilateral lesion that affects most of the seminiferous tubules to varying degrees. It can also be an incidental background finding in rats and mice of any age, but the incidence increases with age (G.Wilson and K. Y. Cimon "Nonneoplastic Lesion Atlas, Uterus, Testis, Germ cell -Degeneration", 2015). (10) Lymphocyte apoptosis is characterized by cell shrinkage, nuclear pyknosis and fragmentation with apoptotic bodies. This type of cell death normally occurs within the germinal centres of secondary follicles where it is an important homeostatic mechanism (Hobbie K, Elmore SA and Kolenda-Roberts HM. 2015. Lymph Node -Apoptosis, Lymphocyte. In: National Toxicology Program Nonneoplastic Lesion Atlas).

Micro Observations
Marrow smear: Examination of the smear showed representative cells from the myeloid and erythroid series. Occasional cells from the lymphoid series. Occasional and unremarkable megakaryoblasts.
Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), numerous white blood cells including lymphocytes, segmented neutrophils, monocytes and platelets (clumps). No discernible morphological changes or detectable parasites.
Micromorphological changes-Stomach: limiting ridge shows focal epithelial hyperplasia, a nearby small submucosal aggregate of cellular debris (possibly mineralisation) and cluster of mononuclear infiltrates (71177), likely to be incidental findings -Pathology to comment-Small intestine: mucosa shows prominent clusters of apoptotic cells (71177,71164,71162), significance unknown -Pathology to comment-Large intestine: numerous intraluminal protozoan parasites (71162), considered an incidental finding Kidneys: few small foci of perivascular lymphocytic inflammation (71169), considered an age related finding Salivary glands: mild multifocal perivascular lymphocytic inflammation (71172, 71173, 71176), considered an age related finding Salivary glands: acini cells of the submandibular gland appear less prominent (71172,71173,71176), significance unknown -Pathology to comment-Lungs: few small foci of perivascular lymphocytic inflammation (71166, 71180), considered an incidental finding Thymus: a small cyst can be seen within the cortex (71166), a common finding in the mouse Heart: mild myxomatous valvular changes (thickened leaflets) (71180, 71181), considered an age related finding Testes/Epididymes Section shows typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. Typical interstitial Leydig cells. Section also shows unremarkable vas deferens with typical intraluminal sperm. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa. Multiple levels through the head demonstrate dermal appendages, nasal cavity, oral cavity, teeth and tongue including muscle bundles. Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (71183). The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (71185-71187). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (71186-71187). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (71186-71187).

Sternum
For further evaluation of bone marrow: Section shows hematopoietic tissue islands surrounded by vascular sinuses interspersed within a meshwork of trabecular bone. The bone marrow morphology demonstrated typical myeloid features including conspicuous megakaryoblasts and lymphoid features.
Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), numerous white blood cells including lymphocytes, segmented neutrophils, monocytes and platelets (clumps). No discernible morphological changes or detectable parasites.
Micromorphological changes-Testes: bilateral, mild focal testicular degeneration (71246), considered an age related finding Stomach: of the glandular portion, small focus of accumulated submucosal neutrophils (71244), considered an incidental finding Small intestine: prominent clusters of apoptotic cells (71244, 71231), significance unknown -Pathology to comment-Large intestine: numerous intraluminal protozoan parasites (71229), considered an incidental finding Kidneys: bilateral, mild multifocal perivascular lymphocytic inflammation (71236), considered an age related finding Salivary glands: of the submandibular gland, mild multifocal perivascular lymphocytic inflammation (71239, 71240), considered an age related finding Salivary gland: acini cells of the submandibular gland appear less prominent (71239, 71240), significance unknown -Pathology to comment-Head: duct adjacent the nasal turbinate shows mild epithelial hyperplasia with intraluminal content admix cellular debris and red blood cells (71249, 71250), considered an incidental finding Head: of the bottom lip, few hair follicles show neutrophilic inflammation (71248), considered an incidental finding Testes/Epididymes Section shows mostly typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. Typical interstitial Leydig cells. Bilateral, mild focal testicular degeneration, considered an age related finding. Section also shows unremarkable vas deferens with typical intraluminal sperm. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa. Sections show a cortex, medulla, and papilla. There is a uniform distribution of glomeruli and accompanying nephron components and the micromorphology of the tubules is unremarkable. Bilateral, mild multifocal perivascular lymphocytic inflammation, considered an age related finding. Sections also include renal lymph nodes with typical reactive nodal histology and mild sinus histiocytosis.  Multiple levels through the head demonstrate dermal appendages, nasal cavity, oral cavity, teeth and tongue including muscle bundles. Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (71226). The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (71247-71250). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (71247-71250). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (71249-71250).
Duct adjacent the nasal turbinate shows mild epithelial hyperplasia with intraluminal content admix cellular debris and red blood cells. Nearby reactive submucosal lymphoid tissue (71249,71250 Marrow smear: Examination of the smear showed representative cells from the myeloid and erythroid series. Occasional cells from the lymphoid series. Occasional and unremarkable megakaryoblasts. Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), numerous white blood cells including lymphocytes, segmented neutrophils, monocytes and platelets (clumps). No discernible morphological changes or detectable parasites.
Micromorphological changes-Testis: one tubule shows features of testicular degeneration (71210), considered an age related finding Small intestine: prominent clusters of apoptotic cells (71208, 71195), significance unknown -Pathology to comment-Large intestine: numerous intraluminal protozoan parasites (71193), considered an incidental finding Kidney: a single dilated protein cast within the medulla (71200), considered an incidental finding Salivary glands: acini cells of the submandibular gland appear less prominent with scattered clusters of mononuclear cellular infiltrates (71203), significance unknown -Pathology to comment-Lungs: small focus of intra-alveolar oedema (71197, 71211, 71212), considered an incidental finding Heart: mild myxomatous valvular changes (thickened leaflets) (71211), considered to be an age related finding Heart: small focal mineralisation (calcification) of the aortic wall (71211), considered to be an incidental finding -Pathology to comment-Skin: several foci of mild epidermal hyperplasia with local neutrophilic inflammation (71205), considered an incidental finding Head: mild inflammation of one hair follicle in the upper lip (71216), considered an incidental finding Testes/Epididymes Section shows mostly typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. Typical interstitial Leydig cells. Section also shows unremarkable vas deferens with typical intraluminal sperm. One tubule shows features of testicular degeneration, considered an age related finding. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa. Salivary glands and Regional lymph nodes Section shows unremarkable sublingual and parotid glands. Acini cells of the submandibular gland appear less prominent with scattered clusters of mononuclear cellular infiltrates. The regional lymph nodes display mild follicular hyperplasia with germinal centre formation, indicating a reactive state. Sections of brain stained with Haematoxylin and Eosin, Luxol Fast Blue appear symmetrical with no ventricular dilation observed, unremarkable meninges and typical lamination. The cerebellum appears symmetrical with typical architecture and Purkinje cells. There was no obvious neuronal loss and the myelination appears normal.  Head harvested for evaluation of auditory and vestibular structures Hind leg-Bone marrow smear Ileal region felt rigid.

Micro Observations
Marrow smear: Examination of the smear showed representative cells from the myeloid and erythroid series. Occasional cells from the lymphoid series. Occasional and unremarkable megakaryoblasts.
Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), occasional white blood cells including lymphocytes, segmented neutrophils, monocytes and platelets (clumps). No discernible morphological changes or detectable parasites.
Micromorphological changes-Skin (penile area): prominent number of melanocytes within the dermis, seemingly accompanied by numerous infiltrates comprised of mast cells and neutrophils (71292, 71294), considered an incidental finding -Pathology to comment-Skin (penile area): mild segmental epithelial hyperplasia with local neutrophilic inflammation at times extending in to the deep dermis (71294, 71297), considered an incidental finding Preputial glands: unilateral, mild scattered mononuclear inflammation accompanied by neutrophils and mast cells (71292, 71294, 71297), considered an incidental finding Stomach: focal epithelial hyperplasia of the limiting ridge (71298), considered an incidental finding -Pathology to comment-Stomach: focal mild submucosal accumulation of neutrophils within the glandular portion (71298), considered an incidental finding Small intestine: disruption of typical mucosal micromorphology with mild enterocyte hyperplasia, most notably of goblet cells, prominent immune cells within the lamina propria, and cytoplasmic vacuolation of apical cells (71298, 71285, 71307) -Pathology to comment-Large intestine: numerous intraluminal protozoan parasites (71307), considered an incidental finding Mesenteric lymph node: pronounced plasmocytosis and scattered neutrophils through the medullary cords and sinuses (71293) -Pathology to comment Kidney: single cystic glomerulus adjacent mild perivascular lymphocytic inflammation, considered an incidental finding (71290) Lung: mild focal intra-alveolar oedema (71287, 71301, 71302), considered an incidental finding Thymus: a small pair of cysts can be seen within the medulla (71301), a common finding in the mouse Heart: mild myxomatous valvular changes (thickened leaflets) (71302), considered an age related finding Skin: mild focal epithelial hyperplasia with local inflammation of the superficial dermis (71295), considered an incidental finding Hind leg: small aggregate of lymphoid and red blood cells between the thigh muscle bundles (71288), considered an incidental finding Head: small cluster of hair follicles adjacent the oral mucosa show neutrophilic inflammation (71311), considered an incidental finding Testes/Epididymes Section shows typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. Typical interstitial Leydig cells. Section also shows unremarkable vas deferens with typical intraluminal sperm. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa. Section shows unremarkable fore and glandular portions of the stomach. Mild focal epithelial hyperplasia of the limiting ridge, and mild focal submucosal accumulation of neutrophils within the glandular portion, considered to be incidental findings. Section also includes pyloric sphincter and duodenal bulb. Eyes/Harderian glands Section shows eyes with unremarkable retina, cornea, iris, ciliary body, lens, sclera and choroid. Typical branched tubuloalveolar formation of the Harderian gland. Section also includes portion of extraocular muscles. (71286) No lesions of significance Brain Sections were prepared from the standard levels of the brain: Level I forebrain: including cortex, corpus callosum, caudate putamen and lateral ventricles (Bregma -0.10mm) Level II midbrain: including the hippocampus, thalamus, hypothalamus and lateral and third ventricles (Bregma -1.58mm) Level III hindbrain: includes the cerebellum, pons and fourth ventricle (Bregma N/A) Sections of brain stained with Haematoxylin and Eosin, Luxol Fast Blue appear symmetrical with no ventricular dilation observed, unremarkable meninges and typical lamination. The cerebellum appears symmetrical with typical architecture and Purkinje cells. There was no obvious neuronal loss and the myelination appears normal. Multiple levels through the head demonstrate dermal appendages, nasal cavity, oral cavity, teeth and tongue including muscle bundles. Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (71303). The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (71309-71311). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (71309-71311). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (71310-71311).
Small cluster of hair follicles adjacent the oral mucosa show neutrophilic inflammation (71311), considered an incidental finding.

Sternum
For further evaluation of bone marrow: Section shows hematopoietic tissue islands surrounded by vascular sinuses interspersed within a meshwork of trabecular bone. The bone marrow morphology demonstrated typical myeloid features including conspicuous megakaryoblasts and lymphoid features. Marrow smear: Examination of the smear showed representative cells from the myeloid and erythroid series. Occasional cells from the lymphoid series. Occasional and unremarkable megakaryoblasts.
Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), occasional white blood cells including lymphocytes, segmented neutrophils, monocytes and platelets (clumps). No discernible morphological changes or detectable parasites.
Micromorphological changes-Preputial glands: scattered perivascular neutrophils (71264), considered an incidental finding Small intestine: disruption of typical mucosal micromorphology with mild enterocyte hyperplasia, most notably of goblet cells, prominent immune cells within the lamina propria, and cytoplasmic vacuolation of apical cells (71257, 71255) -Pathology to comment-Large intestine: numerous intraluminal protozoan parasites (71255), considered an incidental finding Mesenteric lymph node: medullary cords and sinuses occupied by pronounced plasmocytosis and scattered neutrophils (71255, 71265, 71266, 71269) -Pathology to comment-Thymus: small cyst within the cortex (71273), considered an incidental finding Heart: mild myxomatous valvular changes (thickened leaflets) (71273, 71274), considered an age related finding Head: neutrophilic inflammation of a single hair follicle adjacent the oral cavity (71280), considered an incidental finding Sternum: disruption of typical intersternebral joint micromorphology (71429), likely to be artefactual -Pathology to comment-Testes/Epididymes Section shows typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. Typical interstitial Leydig cells. Section also shows unremarkable vas deferens with typical intraluminal sperm. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa.  Level III hindbrain: includes the cerebellum, pons and fourth ventricle  Sections of brain stained with Haematoxylin and Eosin, Luxol Fast Blue appear symmetrical with no ventricular dilation observed, unremarkable meninges and typical lamination. The cerebellum appears symmetrical with typical architecture and Purkinje cells. There was no obvious neuronal loss and the myelination appears normal. (71254,71282) No lesions of significance

Comments:
Neuropathology to comment

Spinal cord
Representative thoracic and lumbar region of spinal cord, vertebral bone, intervertebral disc, striated muscle, peripheral nerves, brown adipose tissue, and bone marrow. Multiple levels through the head demonstrate dermal appendages, nasal cavity, oral cavity, teeth and tongue including muscle bundles. Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (71276). The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (71278-71280). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (71278-71280). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (71279-71280).
Neutrophilic inflammation of a single hair follicle adjacent the oral cavity (71280) Sternum (71429): appears to be loss of matrix staining in articular cartilage with some loss of ground substance (necrotic), possibly due to physical trauma.
Small intestine (71323): mild acute enteritis with increased numbers of neutrophils and plasma cells in lamina propria and enterocyte hyperplasia.

Summary
In the small intestine of three animals (#629, #634 and #635) there are numerous apoptotic bodies, especially in basal regions of the crypts, with some villous atrophy. These changes resemble a radiomimetic effect such as that associated with irradiation or anti-cancer chemotherapeutic drugs. Of the same animals, the salivary glands seem to have varying degrees of acinar atrophy.
The small intestine of the remaining three animals (#640, #641 and #642) show mild acute enteritis with increased numbers of neutrophils and plasma cells in lamina propria and enterocyte hyperplasia.
For more comments, please see above report.