Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aarssti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aarssti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.
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We thank K. Brown, J. Cook, T. Jucius and A. Kano for technical assistance, and the microinjection core at The Jackson Laboratory for transgenic mouse production. This work was supported by National Institutes of Health grants R01NS42613 to S.L.A., R01GM072528 to K.F., R01CA92577 to P.S., and a Fellowship from the National Foundation for Cancer Research to P.S. S.L.A. is an investigator of the Howard Hughes Medical Institute.
Nature thanks C. Francklyn and M. Justice for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 Identification of Ankrd16 as a genetic modifier of the B6J.Aarssti/sti mutation.
a, Three- to four-month-old Aarssti/sti mice from crosses to inbred strains, and the numbers affected with ataxia and Purkinje cell degeneration. b, Location of Msti relative to microsatellite markers. cM, centimorgan. c, Non-synonymous SNPs in Msti candidate genes. The amino acids and positions are shown at the top of the table with the B6 residue listed first. d, RT–PCR analysis of Ankrd16 transcripts from cerebellar cDNA prepared from C57BL/6J and CAST/Ei mice. Note, the alternative Ankrd16 transcript containing exon 5′ (see Fig. 2c) amplified by primers to exon 4/6 or exon 5/7 was present in cDNA from C57BL/6J but not CAST/Ei mice, whereas the alternative transcript detected by exon1/3 primers was present in cDNA from both strains. n = 2 biological replicates. e, RT–PCR analysis of Ankrd16 transcripts from cerebellar cDNA prepared from C57BL/6J, CAST/Ei, Aarssti/sti and MstiCAST/CASTAarssti/sti mice. Note, the alternative Ankrd16 transcript containing exon 5′ (see Fig. 2c) amplified by exon 5/7 or 3/5′ primers was present in cDNA from C57BL/6J and Aarssti/sti, but absent in the presence of CAST/Ei-derived Msti (CAST/Ei and MstiCAST/CASTAarssti/sti mice). n = 3 biological replicates. f, Sequence of the SNP-containing region in intron 5 of Ankrd16. Upper-case letters indicate novel exon 5′ and lower-case letters indicate intron 5. The SNP in non-rescuing strains is shown in red. g, Western blotting analysis of ANKRD16 from cerebellar lysates. Note that the expression of ANKRD16 is reduced in C57BL/6J and Aarssti/sti mice relative to mice with CAST/Ei-derived Msti (CAST/Ei and MstiCAST/CASTAarssti/sti mice) (mean ± s.d., n = 3, one-way ANOVA (Tukey correction), ****P ≤ 0.0001). h, Protein levels of AlaRS and ANKRD16 were determined by western blotting using various mouse tissues from C57BL/6J and B6 congenic mice heterozygous for the Msti region (MstiCAST/B6). GAPDH is included as a loading control. Note the increase in ANKRD16 levels in MstiCAST/B6 tissues, whereas AlaRS levels do not change between genotypes. n = 2 biological replicates for all tissues, n = 4 for neuronal tissues. i, PCR results of genomic DNA from B6 transgenic mouse lines Tg25L9-19 and Tg25L9-46, which carry the CAST/Ei BAC. Polymorphic markers, which differentiate between C57BL/6J and CAST/Ei were used as shown. n = 5 biological replicates. j, Amino acid sequence comparison of ANKRD16 from various species with the C57BL/6J strain shown. Non-synonymous SNPs distinguishing CAST/Ei and C57BL/6J are shown in yellow and serinylated lysines are shown in red. Source Data
a, Peptide/spectral counts of proteins co-immunoprecipitated from transgenic Ankrd16-Myc (see diagram) but not detected in non-transgenic liver tissue. n = 1 experiment. b, HEK293T cells were transiently co-transfected with Myc-tagged constructs for mouse Aars, AarsA734E, the Aars aminoacylation domain (AAD) and Flag-tagged Ankrd16. Co-IP experiments were performed with ANKRD16–Flag as the bait protein. n = 3 independent experiments. c, Reciprocal co-IP experiments were performed by transiently co-transfecting HEK293T cells with HA-tagged constructs for mouse Aars or AarsA734E and the Flag-tagged Ankrd16. HA-AlaRS proteins were used as bait for pull down. n = 3 independent experiments. d, Domain structure of mouse ANKRD16 and ANKRD29. HEK293T cells were transiently transfected with Flag-tagged constructs for mouse Ankrd16, Ankrd29 and the HA-epitope tagged Aars. Co-IP experiments were performed with HA-AlaRS as bait protein. n = 2 independent experiments. e, Various domain protein products of AlaRS (human) as indicated were bacterially expressed, purified, and incubated with GST–ANKRD16. GST-pull down products and input were immunoblotted with anti-His or anti-GST antibodies. Asterisks indicate protein degradation products. n = 2 independent experiments. f, Binding dynamics were determined between mouse wild-type or mutant AlaRS and mouse wild-type or mutant ANKRD16 using switchSENSE (mean ± s.d.; n = 3).
a, Preassembled ternary complex containing either Ser-tRNAAla or Ala-tRNAAla was mixed with 70S initiation complex programmed with codon GCU in the A site, aliquots were transferred at various times to a quenching solution (0.5 M KOH), and products were resolved by eTLC. n = 1 experiment. b, Incubation of full-length AlaRS with preassembled EF-Tu⋅GTP⋅Ser-tRNAAla prevents fMet-Ser formation. EF-Tu⋅GTP⋅Ser-tRNAAla ternary complex and 70S initiation complex were each preassembled. In reaction scheme 1, AlaRS was incubated with EF-Tu⋅GTP⋅Ser-tRNAAla for 15 min, 70S initiation complex was added, and aliquots were removed at various times for eTLC analysis. By contrast, in reaction scheme 2, AlaRS was incubated with the 70S initiation complex for 15 min, followed by addition of EF-Tu⋅GTP⋅Ser-tRNAAla, and aliquots were removed at various times for eTLC analysis. n = 2 independent experiments. c, Deacylated tRNAAla was mixed with AlaRS, serine, ATP and all other components to form the ternary complex, aliquots were transferred at various time points to the 70S initiation complex, and dipeptide products were resolved by eTLC. t = 0 indicates control reactions in the absence of AlaRS; * indicates oxidized fMet. Note, without alanine supplementation (b and c), trace amounts of fMet-Ala are detected, probably due to AlaRS-bound alanyl-AMP during protein purification. n = 2 independent experiments. d, Deacylation of [3H]Ser-tRNAAla by mouse wild-type AlaRS or AlaRS(A734E) in the presence or in the absence of ANKRD16 (mean ± s.d., n = 2, one-phase decay model. R2 values are as follows: wild-type AlaRS, 0.9892; AlaRS(A734E), 0.991; wild-type AlaRS + ANKRD16 = 0.9872; AlaRS(A734E) + ANKRD16, 0.9902; ANKRD16, 0.7992). e, Percentage of EF-Tu retained on the filter membrane upon the addition of various components as indicated (mean ± s.d.; n = 2). f, ATP-pyrophosphate exchange by mouse AlaRS(A734E) in the presence or in the absence of ANKRD16 (mean ± s.d., n = 2, Michaelis–Menten model. R2 values: AlaRS(A734E), 0.9763; AlaRS(A734E) + ANKRD16, 0.9874. g, Aminoacylation of tRNAAla with alanine by mouse AlaRS(A734E) in the presence or in the absence of ANKRD16 (mean ± s.d., n = 2, Michaelis–Menten model. R2 values: AlaRS(A734E), 0.9879; AlaRS(A734E) + ANKRD16, 0.9846. Source Data
a, tRNA-independent ATPase activity of mouse wild-type AlaRS for serine or alanine (mean ± s.d., n = 3, Michaelis–Menten model. R2 for serine, 0.9926; for alanine, 0.8430. b, tRNA-independent ATPase activity of mouse wild-type AlaRS or AlaRS(A734E) for serine in the presence of ANKRD16, ANKRD29, or ANKRD16(3×R) (mean ± s.d., Michaelis–Menten model. R2 values are as follows: wild-type AlaRS, 0.9926; AlaRS(A734E), 0.9899; AlaRS(A734E) + ANKRD16, 0.9918; AlaRS(A734E) + ANKRD29, 0.9939; AlaRS(A734E) + ANKRD16(3×R), 0.9841. For wild-type AlaRS, AlaRS(A734E) and AlaRS(A734E) + ANKRD29, n = 3; for AlaRS(A734E) + ANKRD16 and AlaRS(A734E) + ANKRD16(3×R), n = 4. c, TLC analysis of ATPase activity of AlaRS(A734E) against serine in the absence and in the presence of ANKRD16. n = 3 independent experiments. d, Experimental scheme showing how data were generated for e, f, g and h. e, Acylation reactions with radioactive alanine were performed as described above to determine alanine transfer (mean ± s.d., n = 2, Michaelis–Menten model. R2 values: AlaRS(A734E) + tRNA + ANKRD16, 0.9611; AlaRS(A734E) + tRNA + buffer, 0.9641. f, Misacylation of tRNAAla with radioactive serine by mouse AlaRS(A734E) in the presence or in the absence (buffer) of ANKRD16. After misacylation, reactions were subjected to TCA precipitation of RNA (Ser-tRNAAla) and protein under acidic conditions to maintain Ser-tRNA (mean ± s.d., n = 2, Michaelis–Menten model. R2 values AlaRS(A734E) + tRNA + ANKRD16, 0.9577; AlaRS(A734E) + tRNA + buffer, 0.7841. g, Misacylation reactions using radioactive serine and mouse wild-type AlaRS or AlaRS(A734E) were performed in the presence or in the absence of ANKRD16, ANKRD29, ANKRD16(3×R) or tRNAAla. After TCA precipitation under neutral pH conditions, serine was measured (mean ± s.d., n = 4). Note the higher level of TCA-precipitated serine on tRNA or protein in the presence of ANKRD16. h, Acylation reactions using radioactive alanine and mouse AlaRS(A734E) were performed in the presence or in the absence of ANKRD16 or tRNAAla. After TCA precipitation under neutral pH conditions, alanine was measured (mean ± s.d., n = 4). i, Misacylation reactions using radioactive serine and mouse AlaRS(A734E) were performed in the presence of ANKRD16 either with or without tRNAAla. Reactions were treated with or without Na2CO3 (final concentration of 0.15 M, alkaline pH) for 30 min, followed by TCA precipitation. Precipitated [3H]serine-links were measured and plotted as relative level of serinylation (mean, n = 2). Source Data
a–d, MS/MS spectra of peptides from ANKRD16. Incorporation of serine onto ANKRD16 was observed when serine was misactivated by mouse AlaRS(A734E) (+ATP). Misactivation was not observed in the absence of ATP. MS/MS spectrum of peptides from ANKRD16 with serine linked to positions K102 (b), K135 (c) and K165 (d). a ions (a), b ions (b) and y ions (y) are annotated in green, red and purple, respectively. The triply charged precursor had a mass of 2,153.065 daltons and included carbamidomethyl cysteine. For a–d, n = 1 experiment. e, Secondary structure analysis of mouse ANKRD16 and ANKRD16(3×R). Far-UV circular dichroism spectra of wild-type ANKRD16 (blue) and ANKRD16(3×R) (red) show highly similar CD spectra (mean ± s.d., n = 4). f, Thermal-shift analysis of mouse ANKRD16 (blue) and ANKRD16(3×R) (red) show highly similar thermal stability. n = 2 independent experiments. g, HEK293T cells were transiently co-transfected with Myc-tagged constructs for mouse AlaRS(A734E) and Flag-tagged ANKRD16, ANKRD16(3×R) or ANKRD29. Co-IP experiments were performed with Flag-tagged proteins as the bait protein. Binding affinity was determined by normalizing the immunoprecipitation signal of AlaRS(A734E) to the input signal, in which the interaction of AlaRS(A734E) + ANKRD16 was arbitrarily defined as 1 to determine the relative binding affinity of AlaRS(A734E) + ANKRD16(3×R) (mean ± s.d., n = 3). Source Data
B6.Aarssti/sti embryonic fibroblasts were co-transfected with hrGFP (humanized recombinant GFP, green) and either ANKRD16–Flag or ANKRD16(3×R)–Flag (n = 4). 12 h post-transfection, serine was added and cells were cultured for 24 h before staining with propidium iodide (PI; red) and Hoechst (blue) to determine cell death. Arrowheads represent PI+GFP+ cells. n = 4 biological replicates. Scale bars, 100 μm.
a, Protein levels of ANKRD16 were determined by western blotting of tissues from B6 mice homozygous for the Msti region (MstiCAST/CAST). n = 3 biological replicates. b, Subcellular analysis of ANKRD16 and AlaRS by cell fractionation of brains from B6.MstiCAST/B6 and Ankrd16−/− mice. Cellular fractions were confirmed using antibodies for histone 3 (nuclear marker), GAPDH (cytosolic marker), COX IV (mitochondrial marker), GRP78 (endoplasmic reticulum marker), and Sec61 beta (rough endoplasmic reticulum marker). n = 2 independent experiments. c, To generate a ubiquitous or conditional loss-of-function allele, loxP sites that flank exon 2 of Ankrd16 were inserted by homologous recombination. Removal of exon 2 results in a frame shift and premature stop codon in exon 3. Ankrd16 was ubiquitously deleted by EIIa Cre-mediated removal of exon 2 and the neo cassette. Flippase (Flp)-mediated excision of the neo cassette was used to generate a conditional loss-of-function Ankrd16 allele. d, Loss of ANKRD16 was verified by western blotting. Protein extracts from Ankrd16−/−, MstiCAST/CAST and C57BL/6J mice were used for comparison purposes and GAPDH was used as a loading control. n = 4 biological replicates. e, The number of embryos or mice of various genotypes from intercrosses of Aarssti/+Ankrd16−/− mice over the total number observed. Representative images of E9.5 embryos. Note, Aarssti/stiAnkrd16−/− embryos are smaller and have failed to turn. E, embryonic day; P, postnatal day. For E8.5 and E9.5, n = 4 litters for each time point; E10.5 and E12.5, n = 2 litters for each time point; P21, n = 7 litters. Scale bars, 500 μm. Source Data
Extended Data Fig. 8 Conditional deletion of Ankrd16 accelerates Purkinje cell loss and causes widespread neurodegeneration in the B6.Aarssti/sti cerebellum.
a, b, Calbindin D-28 immunohistochemistry of sagittal cerebellar sections. c, Cresyl violet-stained sagittal cerebellar sections. Note the presence of interneurons in the molecular layer (ML) of B6.Aarssti/sti cerebellum despite the thinning of the molecular layer as a consequence of Purkinje cell degeneration. By contrast, loss of Ankrd16 in the B6.Aarssti/sti cerebellum results in degeneration of molecular-layer interneurons. GL, granule cell layer; PL, Purkinje cell layer. The number of biological replicates are as follows: for TgPcp2-Cre Ankrd16fl/−Aarssti/sti or En1creAnkrd16fl/−Aarssti/sti: P7, n = 5; P18, n = 4; P21, n = 4; P28, n = 7; P30, n = 5; P42, n = 4; 3 months, n = 5; 7 months, n = 6. For Ankrd16−/−: 7 months, n = 7. For Aarssti/sti and wild-type: 7 months, n = 3. Scale bars, 500 μm (a, b, c), 50 μm (c, higher magnification).
Extended Data Fig. 9 Conditional deletion of Ankrd16 accelerates the formation of protein aggregates in B6.Aarssti/sti mice.
a, Ubiquitin (Ub; red), p62 (green), and Calbindin D-28 (Calb; blue) immunofluorescence on sagittal cerebellar sections. The percentage of aggregate-positive Purkinje cells and Purkinje cells are shown (mean ± s.d., n = 3, multiple t-tests (Holm–Sidak method), *** P = 0.0002466 (Purkinje cells with aggregates, P21), P = 0.0002336 (Purkinje cells with aggregates, P28), P = 0.0003214 (percentage of Purkinje cells, P21), ****P = 7.701787 × 10−5 (percentage of Purkinje cells, P28)). Note that the percentage of Purkinje cells is relative to control C57BL/6J mice. b, Cell type-specific markers (red) and p62 (green) immunofluorescence on sagittal cerebellar sections. Parvalbumin (Parv) was used to identify Purkinje cells and interneurons (stellate and basket cells) in the molecular layer. NeuN was used to distinguish between granule and Golgi cells in the granule cell layer. En1cre Ankrd16fl/− Aarssti/sti: Golgi cell (closed arrow head, p62+NeuN−); granule cell (arrow, p62+/NeuN+); basket or stellate cell (open arrow head, p62+/Parv+). n = 3 biological replicates. c, Ubiquitin (red) and p62 (green) immunofluorescence on sagittal sections of the cortex (layer IV). n = 3 biological replicates. Scale bars, 10 μm (a), 50 μm (b and c, low magnification) and 10 μm (b and c, higher magnification). Source Data
a, A point mutation in the editing domain of AlaRS(A734E) results in editing defects, as indicated by deficits in tRNA-independent ATPase activity, which in turn leads to increased levels of incorrectly aminoacylated Ser-tRNAAla, misincorporation of serine during translation, protein aggregation and cell death. b, Interaction of ANKRD16 with the aminoacylation domain of editing-deficient AlaRS(A734E) stimulates tRNA-independent editing, and misactivated serines are transferred onto ANKRD16. Mitigation of serine misactivation prevents sti-mediated mistranslation, and thereby prevents protein aggregation and cell death.
This file contains the western blots that are labelled with the corresponding figure number and panel letter, antibody staining and the region of the blot that has been cropped. Western blots are provided for the following figures: Fig.2e, Fig. 3a, b and c, Extended Data Fig. 1g and h, Extended Data Fig.2b, c, d, and e, Extended Data Fig. 5g and Extended Data Fig. 7a, b and d.