Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle.

Apobec2 is a member of the activation-induced deaminase/apolipoprotein B mRNA editing enzyme catalytic polypeptide cytidine deaminase family expressed in differentiated skeletal and cardiac muscle. We previously reported that Apobec2 deficiency in mice leads to a shift in muscle fiber type, myopathy, and diminished muscle mass. However, the mechanisms of myopathy caused by Apobec2 deficiency and its physiologic functions are unclear. Here we show that, although Apobec2 localizes to the sarcomeric Z-lines in mouse tissue and cultured myotubes, the sarcomeric structure is not affected in Apobec2-deficient muscle. In contrast, electron microscopy reveals enlarged mitochondria and mitochondria engulfed by autophagic vacuoles, suggesting that Apobec2 deficiency causes mitochondrial defects leading to increased mitophagy in skeletal muscle. Indeed, Apobec2 deficiency results in increased reactive oxygen species generation and depolarized mitochondria, leading to mitophagy as a defensive response. Furthermore, the exercise capacity of Apobec2-/- mice is impaired, implying Apobec2 deficiency results in ongoing muscle dysfunction. The presence of rimmed vacuoles in myofibers from 10-mo-old mice suggests that the chronic muscle damage impairs normal autophagy. We conclude that Apobec2 deficiency causes mitochondrial defects that increase muscle mitophagy, leading to myopathy and atrophy. Our findings demonstrate that Apobec2 is required for mitochondrial homeostasis to maintain normal skeletal muscle function.-Sato, Y., Ohtsubo, H., Nihei, N., Kaneko, T., Sato, Y., Adachi, S.-I., Kondo, S., Nakamura, M., Mizunoya, W., Iida, H., Tatsumi, R., Rada, C., Yoshizawa, F. Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle.

CTNNBL1 facilitates the association of CWC15 with CDC5L and is required to maintain the abundance of the Prp19 spliceosomal complex.

In order to catalyse the splicing of messenger RNA, multiple proteins and RNA components associate and dissociate in a dynamic highly choreographed process. The Prp19 complex is a conserved essential part of the splicing machinery thought to facilitate the conformational changes the spliceosome undergoes during catalysis. Dynamic protein interactions often involve highly disordered regions that are difficult to study by structural methods. Using amine crosslinking and hydrogen-deuterium exchange coupled to mass spectrometry, we describe the architecture of the Prp19 sub-complex that contains CTNNBL1. Deficiency in CTNNBL1 leads to delayed initiation of cell division and embryonic lethality. Here we show that in vitro CTNNBL1 enhances the association of CWC15 and CDC5L, both core Prp19 complex proteins and identify an overlap in the region of CDC5L that binds either CTNNBL1 or CWC15 suggesting the two proteins might exchange places in the complex. Furthermore, in vivo, CTNNBL1 is required to maintain normal levels of the Prp19 complex and to facilitate the interaction of CWC15 with CDC5L. Our results identify a chaperone function for CTNNBL1 within the essential Prp19 complex, a function required to maintain the integrity of the complex and to support efficient splicing.

Interaction between antibody-diversification enzyme AID and spliceosome-associated factor CTNNBL1.

Activation-induced deaminase (AID) deaminates deoxycytidine residues in immunoglobulin genes, triggering antibody diversification. Here, by use of two-hybrid and coimmunoprecipitation assays, we identify CTNNBL1 (also known as NAP) as an AID-specific interactor. Mutants of AID that interfere with CTNNBL1 interaction yield severely diminished hypermutation and class switching. Targeted inactivation of CTNNBL1 in DT40 B cells also considerably diminishes IgV diversification. CTNNBL1 is a widely expressed nuclear protein that associates with the Prp19 complex of the spliceosome, interacting with its CDC5L component. The results, therefore, identify residues in AID involved in its in vivo targeting and suggest they might act through interaction with CTNNBL1, giving possible insight into the linkage between AID recruitment and target-gene transcription.

The cytoplasmic AID complex.

Although AID fulfils its physiological function of diversifying antibody genes in the nucleus, most of the AID protein within the cell is found in a complex located in the cytoplasm. In this review, we summarize what is currently known about this cytoplasmic AID complex. Its size has been estimated to lie between 300 and 500kDa (sedimentation coefficient of 10-11S) and it comprises the abundant protein translation elongation factor 1α (eEF1A) as a major stoichiometric component. We speculate on the possible roles of this complex as well as of chaperones known to interact with AID in regulating the cytosolic retention of AID and its controlled release for import into the nucleus.

Somatic hypermutation at A.T pairs: polymerase error versus dUTP incorporation.

Somatic hypermutation of immunoglobulin genes occurs at both C.G pairs and A.T pairs. Mutations at C.G pairs are created by activation-induced deaminase (AID)-catalysed deamination of C residues to U residues. Mutations at A.T pairs are probably produced during patch repair of the AID-generated U.G lesion, but they occur through an unknown mechanism. Here, we compare the popular suggestion of nucleotide mispairing through polymerase error with an alternative possibility, mutation through incorporation of dUTP (or another non-canonical nucleotide).

Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation.

Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil-DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung(-/-) mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung(-/-) mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung(-/-) mice depends upon the endogenous SMUG1 uracil-DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung(-/-) Smug1(-/-) mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2-dependent mechanism, Ung(-/-) Smug1(-/-) mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung(-/-) background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.

Germline ablation of SMUG1 DNA glycosylase causes loss of 5-hydroxymethyluracil- and UNG-backup uracil-excision activities and increases cancer predisposition of Ung-/-Msh2-/- mice.

Deamination of cytosine (C), 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) occurs spontaneously in mammalian DNA with several hundred deaminations occurring in each cell every day. The resulting potentially mutagenic mispairs of uracil (U), thymine (T) or 5-hydroxymethyluracil (hmU) with guanine (G) are substrates for repair by various DNA glycosylases. Here, we show that targeted inactivation of the mouse Smug1 DNA glycosylase gene is sufficient to ablate nearly all hmU-DNA excision activity as judged by assay of tissue extracts from knockout mice as well as by the resistance of their embryo fibroblasts to 5-hydroxymethyldeoxyuridine toxicity. Inactivation of Smug1 when combined with inactivation of the Ung uracil-DNA glycosylase gene leads to a loss of nearly all detectable uracil excision activity. Thus, SMUG1 is the dominant glycosylase responsible for hmU-excision in mice as well as the major UNG-backup for U-excision. Both Smug1-knockout and Smug1/Ung-double knockout mice breed normally and remain apparently healthy beyond 1 year of age. However, combined deficiency in SMUG1 and UNG exacerbates the cancer predisposition of Msh2(-/-) mice suggesting that when both base excision and mismatch repair pathways are defective, the mutagenic effects of spontaneous cytosine deamination are sufficient to increase cancer incidence but do not preclude mouse development.

Cytoplasmic activation-induced cytidine deaminase (AID) exists in stoichiometric complex with translation elongation factor 1α (eEF1A).

Activation-induced cytidine deaminase (AID) is a B lymphocyte-specific DNA deaminase that acts on the Ig loci to trigger antibody gene diversification. Most AID, however, is retained in the cytoplasm and its nuclear abundance is carefully regulated because off-target action of AID leads to cancer. The nature of the cytosolic AID complex and the mechanisms regulating its release from the cytoplasm and import into the nucleus remain unknown. Here, we show that cytosolic AID in DT40 B cells is part of an 11S complex and, using an endogenously tagged AID protein to avoid overexpression artifacts, that it is bound in good stoichiometry to the translation elongation factor 1 alpha (eEF1A). The AID/eEF1A interaction is recapitulated in transfected cells and depends on the C-terminal domain of eEF1A (which is not responsible for GTP or tRNA binding). The eEF1A interaction is destroyed by mutations in AID that affect its cytosolic retention. These results suggest that eEF1A is a cytosolic retention factor for AID and extend on the multiple moonlighting functions of eEF1A.

Somatic hypermutation: activation-induced deaminase for C/G followed by polymerase eta for A/T.

Somatic hypermutation (SHM) introduces nucleotide substitutions into immunoglobulin variable (Ig V) region genes at all four bases, but the mutations at C/G and A/T pairs are achieved by distinct mechanisms. Mutations at C/G pairs are a direct consequence of the C-->U deamination catalyzed by activation-induced deaminase (AID). Mutations at A/T pairs, however, require a second mutagenic process that occurs during patch repair of the AID-generated U/G mismatch. Several DNA polymerases have been proposed to play a role in SHM, but accumulating evidence indicates that the mutations at A/T are overwhelmingly achieved by recruitment of DNA polymerase eta.

Erratum: Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues.

[This corrects the article DOI: 10.1016/j.omtm.2019.05.012.].

CTNNBL1 is a novel nuclear localization sequence-binding protein that recognizes RNA-splicing factors CDC5L and Prp31.

Nuclear proteins typically contain short stretches of basic amino acids (nuclear localization sequences; NLSs) that bind karyopherin α family members, directing nuclear import. Here, we identify CTNNBL1 (catenin-ß-like 1), an armadillo motif-containing nuclear protein that exhibits no detectable primary sequence homology to karyopherin α, as a novel, selective NLS-binding protein. CTNNBL1 (a single-copy gene conserved from fission yeast to man) was previously found associated with Prp19-containing RNA-splicing complexes as well as with the antibody-diversifying enzyme AID. We find that CTNNBL1 association with the Prp19 complex is mediated by recognition of the NLS of the CDC5L component of the complex and show that CTNNBL1 also interacts with Prp31 (another U4/U6.U5 tri-snRNP-associated splicing factor) through its NLS. As with karyopherin αs, CTNNBL1 binds NLSs via its armadillo (ARM) domain, but displays a separate, more selective NLS binding specificity. Furthermore, the CTNNBL1/AID interaction depends on amino acids forming the AID conformational NLS with CTNNBL1-deficient cells showing a partial defect in AID nuclear accumulation. However, in further contrast to karyopherin αs, the CTNNBL1 N-terminal region itself binds karyopherin αs (rather than karyopherin ß), suggesting a function divergent from canonical nuclear transport. Thus, CTNNBL1 is a novel NLS-binding protein, distinct from karyopherin αs, with the results suggesting a possible role in the selective intranuclear targeting or interactions of some splicing-associated complexes.

The in vivo pattern of AID targeting to immunoglobulin switch regions deduced from mutation spectra in msh2-/- ung-/- mice.

Immunoglobulin (Ig) class switching is initiated by deamination of C-->U within the immunoglobulin heavy chain locus, catalyzed by activation-induced deaminase (AID). In the absence of uracil-DNA glycosylase (UNG) and the homologue of bacterial MutS (MSH)-2 mismatch recognition protein, the resultant U:G lesions are not processed into switching events but are fixed by replication allowing sites of AID-catalyzed deamination to be identified by the resulting C-->T mutations. We find that AID targets cytosines in both donor and acceptor switch regions (S regions) with the deamination domains initiating approximately 150 nucleotides 3' of the I exon start sites and extending over several kilobases (the IgH intronic enhancer is spared). Culturing B cells with interleukin 4 or interferon gamma specifically enhanced deamination around Sgamma1 and Sgamma2a, respectively. Mutation spectra suggest that, in the absence of UNG and MSH2, AID may occasionally act at the mu switch region in an apparently processive manner, but there is no marked preference for targeting of the transcribed versus nontranscribed strand (even in areas capable of R loop formation). The data are consistent with switch recombination being triggered by transcription-associated, strand-symmetric AID-mediated deamination at both donor and acceptor S regions with cytokines directing isotype specificity by potentiating AID recruitment to the relevant acceptor S region.

The dependence of Ig class-switching on the nuclear export sequence of AID likely reflects interaction with factors additional to Crm1 exportin.

Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that - beyond a requirement for threshold Crm1 binding - there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1.

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence.

The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR.

Deficiency in APOBEC2 leads to a shift in muscle fiber type, diminished body mass, and myopathy.

The apoB RNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of proteins includes APOBEC1, APOBEC3, and activation-induced deaminase, all of which are zinc-dependent cytidine deaminases active on polynucleotides and involved in RNA editing or DNA mutation. In contrast, the biochemical and physiological functions of APOBEC2, a muscle-specific member of the family, are unknown, although it has been speculated, like APOBEC1, to be an RNA-editing enzyme. Here, we show that, although expressed widely in striated muscle (with levels peaking late during myoblast differentiation), APOBEC2 is preferentially associated with slow-twitch muscle, with its abundance being considerably greater in soleus compared with gastrocnemius muscle and, within soleus muscle, in slow as opposed to fast muscle fibers. Its abundance also decreases following muscle denervation. We further show that APOBEC2-deficient mice harbor a markedly increased ratio of slow to fast fibers in soleus muscle and exhibit an approximately 15-20% reduction in body mass from birth onwards, with elderly mutant animals revealing clear histological evidence of a mild myopathy. Thus, APOBEC2 is essential for normal muscle development and maintenance of fiber-type ratios; although its molecular function remains to be identified, biochemical analyses do not especially argue for any role in RNA editing.

In memoriam: Jefferson Foote.

In a scientific career that spanned over three decades, Dr. Jeff Foote made seminal contributions to antibody humanization and the biophysical aspects of antibody recognition. In this Perspective, we discuss his life and work.

Erratum: The uracil-DNA glycosylase UNG protects the fitness of normal and cancer B cells expressing AID.

[This corrects the article DOI: 10.1093/narcan/zcaa019.].

The AKV murine leukemia virus is restricted and hypermutated by mouse APOBEC3.

APOBEC3 proteins are potent restriction factors against retroviral infection in primates. This restriction is accompanied by hypermutations in the retroviral genome that are attributable to the cytidine deaminase activity of the APOBEC3 proteins. Studies of nucleotide sequence diversity among endogenous gammaretroviruses suggest that the evolution of endogenous retroelements could have been shaped by the mutagenic cytidine deaminase activity of APOBEC3. In mice, however, APOBEC3 appears to restrict exogenous murine retroviruses in the absence of detectable levels of deamination. AKV is an endogenous retrovirus that is involved in causing a high incidence of thymic lymphoma in AKR mice. A comparative analysis of several mouse strains revealed a relatively low level of APOBEC3 expression in AKR mice. Here we show that endogenous mouse APOBEC3 restricts AKV infection and that this restriction likely reflects polymorphisms affecting APOBEC3 abundance rather than differences in the APOBEC3 isoforms expressed. We also observe that restriction of AKV by APOBEC3 is accompanied by G-->A hypermutations in the viral genome. Our findings demonstrate that APOBEC3 acts as a restriction factor in rodents affecting the strain tropism of AKV, and they provide good support for the proposal that APOBEC3-mediated hypermutation contributed to the evolution of endogenous rodent retroviral genomes.