Fig 1.
Expression of RBM-26 is disrupted by P80L and L13V missense mutations.
(A) Putative protein domains in human RBM27, and C. elegans RBM-26 proteins predicted by NCBI’s CD-Search. CCCH-type Zn Finger domains (Green Rectangles) and RNA Recognition Motifs (Gold Pentagons) were predicted with high confidence by NCBI’s CD-Search and SMART database. PWI superfamily (Blue Hexagon) was predicted by NCBI’s CD-Search with high confidence. Red arrows indicate de novo missense variants identified in patients with autism or other neurodevelopmental disorders. (B) Clustal Omega alignment of the N-terminus of the human RBM27 and C. elegans RBM-26 proteins. Two de novo RBM27 variants, L13V and P79L (red arrows), were found in regions conserved in C. elegans RBM-26. (C) Representative western blot of 3 biological replicates showing expression of RBM-26::3XFLAG, RBM-26 P80L::3XFLAG, and RBM-26 L13V::3XFLAG proteins; 20 μg of total protein lysate was loaded per well and specific proteins were detected with an anti-FLAG antibody and enhanced chemiluminescence. Protein loading was quantified by No-Stain Protein Labeling Reagent. (D) Densitometric quantification of protein bands. Statistical significance was analyzed by one-way ANOVA with multiple comparison testing (*p < 0.05, ***p < 0.0001). (E) Example of endogenously tagged RBM-26 in wild type at the L3 stage. (F) Example of endogenously tagged RBM-26 in rbm-26(P80L) mutant at the L3 stage. RBM-26 was endogenously tagged with Scarlet using CRISPR to generate the rbm-26(syb2552[rbm-26::Scarlet::AID]) allele. CRISPR was used to insert the P80L mutation into rbm-26 (syb2552) to create the rbm-26(cue48[rbm-26(P80L)] syb2552[rbm-26::Scarlet::AID]) allele. Underlying data can be found in S1 Data.
Fig 2.
Mutations in rbm-26 cause axon degeneration and axon overlap defects.
(A) Example of a normal PLM axon, where the PLM axon does not overlap with the ALM axon (B) Diagram of the touch receptor neurons. (C) Example of an axon overlap defect (PLM/ALM overlap phenotype) in rbm-26 (null) L3 worms. Axon beading can also be seen (asterisk). (D) Quantification of axon overlap defects in various stages. (E) Example of axon beading phenotype (asterisks) in an rbm-26(null) mutant. (F) Quantification of beading phenotype at various stages. (G) Example of the axonal break phenotype in the PLM of a 2-day-old adult rbm-26 (P80L) mutant. (H) Quantification of the axonal break phenotype at various stages. Asterisks indicate statistically significant difference relative to wild type, Z-test for proportions (*** p < 0.0001, ** p < 0.01, and * p < 0.05), and error bars represent the standard error of the proportion. Scale bars are 10 μm. Axons are visualized with the muIs32 (Pmec-7::gfp) transgene. Stages are labeled with L1 (first larval stage), L2 (second larval stage), L3 (third larval stage), L4 (fourth larval stage), 1-Day Adult and 2-Day Adult. Alleles: rbm-26(null) is rbm-26(gk910); rbm-26(P80L) is rbm-26(cue23); rbm-26(L13V) is rbm-26(cue34). Underlying data can be found in S1 Data.
Fig 3.
RBM-26 is expressed in the PLM and functions within neurons to protect against axon degeneration and prevent axon overlap defects.
(A) PLM is identified by GFP expression driven by the muIs32 transgene that encodes Pmec-7::gfp. (B) Expression of endogenously tagged RBM-26. The endogenously tagged allele of RBM-26 is rbm-26 [syb2552(rbm-26::Scarlet::AID)]. A and B are merged in (C). (D, E) Neuron-specific degradation of RBM-26::Scarlet::AID causes the PLM/ALM overlap phenotype and axon beading. However, degradation in hypodermis, muscle, and intestine do not cause these phenotypes. Control strain is rbm-26::Scarlet::AID. (F, G) The PLM/ALM overlap and the beading phenotypes caused by mutations in rbm-26 are rescued by the expression of the cueSi52(Pmec-7::rbm-26::scarlet::unc-54 3’ UTR) transgene, which expresses RBM-26 in PLM and other touch receptor neurons. Asterisks indicate statistically significant difference, Z-test for proportions (*p < 0.05, ***p < 0.001), and error bars represent the standard error of the proportion. n = 150 for D and E and 200 in F and G. Touch receptor neurons were visualized in D–G with the muIs32 transgene. Underlying data can be found in S1 Data.
Fig 4.
Loss of RBM-26 function causes a reduction in the density of mitochondria in the PLM axon.
(A–E) RBM-26 loss of function reduces the number of mitochondria in the PLM axon. (A, B) Example of mitochondria (arrowheads) in an L3 wild-type PLM. (C, D) Example of mitochondria in rbm-26(null) L3 PLM. PLM axons are visualized with the muIs32 transgene that encodes Pmec-7::gfp and mitochondria are visualized with jsIs1073 transgene that encodes Pmec-7::mito:RFP. B and D show mitochondria only. Images depict the middle of the PLM axon. Scale bars are 10 μm. (E) Quantification of the density of mitochondria (number of mitochondria per 100 μm of PLM axon) in axons at the L3 stage (n ≥ 25). Statistical significance was analyzed by one-way ANOVA with a Tukey post hoc test, **p < 0.01. (F) Quantification of the density of mitochondria (number of mitochondria per 100 μm) in L1, L2, and L3 PLM axons; 20 PLMs were analyzed for L1 and L2, and 25 PLMs were analyzed for L3. Statistical significance was analyzed by two-tailed Student’s t test. The reduction in the number of mitochondria in PLM axons in the rbm-26 mutants was limited to the proximal PLM axon (50 μm closest to the cell body) (G) and was not observed in the distal PLM axon (50 μm closest to the axon tip) (H). A total of 20 PLMs were observed for each genotype in G and H. Statistical significance was analyzed by one-way ANOVA with a Tukey post hoc test, ** p < 0.01, ns = not significant. Error bars in E–H are the standard error of mean. Alleles: rbm-26(null) is rbm-26(gk910); rbm-26(P80L) is rbm-26(cue23); rbm-26(L13V) is rbm-26(cue34). Underlying data can be found in S1 Data.
Fig 5.
Loss of RBM-26 causes mitochondria dysfunction in PLM.
RBM-26 loss of function causes excess oxidative activity in mitochondria. To detect oxidative activity in mitochondria, we used mitoTimer, which is a green fluorescent protein when newly synthesized but shifts to red fluorescence when oxidized. (A-A”) Example of mitoTimer expression in an L3 wild-type PLM cell body. (B-B”) Example of mitoTimer expression in an L3 rbm-26(null) mutant PLM cell body. Scale bars are 4 μm. (C–E) Quantification of the ratio of red to green mitoTimer expression in wt and rbm-26 mutant PLM cell bodies in different larval stages. Statistical significance was analyzed by one-way ANOVA with a Tukey post hoc test (*p < 0.05, **p < 0.01 and ns = not significant); n = 30 for C and E, and 21 for D. Error bars represent the standard error of mean. The mitoTimer transgene is cueSi35 IV (Pmec-7::mitoTimer::tbb-2 3′ UTR). Alleles: rbm-26(null) is rbm-26(gk910); rbm-26(P80L) is rbm-26(cue23); rbm-26(L13V) is rbm-26(cue34). Underlying data can be found in S1 Data.
Fig 6.
RBM-26 binds to the mals-1 mRNA and negatively regulates its expression.
(A) RNA interactors of RBM-26 as identified by UV-RIPseq (see Methods for details). Data generated from 3 biological replicates. (B) Loss of RBM-26 function increases expression of the mals-1 mRNA. Absolute levels of mals-1 mRNA quantified using real-time RT-PCR (see Methods for details). Graph represents averages of 3 biological replicates. Error bars are standard deviations. Asterisks indicate statistically significant difference relative to wild type, two-tailed Student’s t test (***p < 0.0001). (C) Loss of RBM-26 function causes an increase in MALS-1 protein expression. Wild-type worms are mals-1(cue37) that expresses MALS-1::3X FLAG. CRISPR was used to insert P80L and L13V mutations into the mals-1(cue37) chromosome generating rbm-26(cue40) mals-1(cue37) and rbm-26(cue49) mals-1(cue37), respectively; 20 μg of total protein lysate from a mixed stage population was loaded per well in the top panel and specific proteins were detected with an anti-FLAG antibody and enhanced chemiluminescence. Loading was assessed with No-Stain protein labeling reagent, and 40 μg of total protein lysate was loaded for wild-type and rbm-26(cue40) mals-1(cue37) mutants in bottom panels. RIPA buffer was used to prepare lysates used in the top panel and HNM buffer was to prepare lysates used in bottom panel (see Methods for buffer details). A nonspecific band can be seen in rbm-26 (L13V) sample in the bottom panel possibly due to buffer conditions, overloading and overexposure. (D) Densitometric quantification of MALS-1 protein levels in western blots. Data are expressed as mean ± SEM from 3 independent experiments. Asterisks indicate statistically significant difference relative to wild type, two-tailed Student’s t test (**p < 0.01,*p < 0.05). Underlying data can be found in S1 Data.
Fig 7.
Overexpression of MALS-1 reduces mitochondria in the PLM axon and causes axon degeneration and axon overlap defects.
(A–C) Overexpressed MALS-1::Scarlet protein colocalizes with mitochondria. (A) Mitochondria visualized with the jsIs609 transgene, which expresses a mitochondria-targeted GFP (mitoGFP) in the PLM neuron. (B) MALS-1::Scarlet expressed by the cueSi36 IV transgene. (C) Merged image showing colocalization of MALS-1::Scarlet with mitoGFP. (D–F) Overexpression of MALS-1 with the Pmec-7::mals-1 transgenes (cueEx53 and cueEx54) causes defects similar to those caused by loss of RBM-26 function. (D) Axon overlap defects caused by MALS-1 overexpression. (E) PLM axon beading phenotype caused by MALS-1 overexpression. (F) Decrease in mitochondria density (number of mitochondria per 100 μm) in the PLM axon caused by MALS-1 overexpression. Asterisks in panels D and E indicate statistically significant difference relative to wild type, Z-test for proportions (***p < 0.0001). For panels D and E, error bars represent the standard error of the proportion. Statistical significance in panel F was analyzed by Student’s t test, ***p < 0.0001 and error bars represent the standard error of mean. n = 100 in D and E, and 20 in F. Underlying data can be found in S1 Data.
Fig 8.
Loss of MALS-1 function suppresses the mitochondrial and axon degeneration phenotypes that are caused by loss of RBM-26 function.
(A) Loss of mals-1 function suppresses the PLM axon beading phenotype caused by the rbm-26 mutations. (B) Loss of mals-1 function suppresses the reduction of mitochondria caused by the rbm-26 mutations. (C) Loss of mals-1 function causes PLM axon termination defect but fails to enhance PLM axon termination defects caused by the rbm-26 mutations. Control (wild type), rbm-26 (cue23[P80L]) and rbm-26 (cue34[L13V]) data in panels A, B, and C have been reused from Figs 2D, 2F and 5C, respectively. The rbm-26(cue40 [P80L]) mals-1(syb6330) double mutant was created by introducing the P80L mutation into the mals-1(syb6330) mutant chromosome. The rbm-26(cue41 [P80L]) mals-1(tm12122) double mutant was created by introducing the P80L mutation into the mals-1(tm12122) mutant chromosome. The rbm-26 (cue47 [L13V]) mals-1(syb6330) double mutant was created by introducing the L13V mutation into the mals-1(syb6330) mutant chromosome. Asterisks in A and C indicate statistically significant difference, Z-test for proportions (*p < 0.05, **p < 0.01, ***p < 0.0001). For panels A and C, error bars represent the standard error of the proportion (n = 200). For panel B, error bars represent the standard error of the mean (n = 25), and statistical significance was analyzed by one-way ANOVA with a Tukey post hoc test (*p < 0.05 and **p < 0.01). (D) Loss of RBM-26 protein expression causes a decrease in mitoribosome expression, which was assayed with the MitoRibo-Tag system consisting of the MRPL-58 mitoribosomal protein tagged at its C-terminus with 3XFLAG (MRPL-58::3XFLAG). This MRPL-58::3XFLAG protein was expressed by the mrpl-58(cue38) allele, which was created by CRISPR. mrpl-58(cue38) rbm-26(cue39) double mutant was created by introducing the P80L mutation into mrpl-58(cue38) by CRISPR. CRISPR was also used to insert a 3X FLAG tag at the C-terminus of MRPL-58 in mals-1(syb6330) mutant to create mrpl-58(cue50) mals-1(syb6330). Finally, rbm-26 (P80L) mutation was inserted into mrpl-58(cue50) mals-1(syb6330) to create rbm-26(cue51 [P80L]) mrpl-58(cue50) mals-1(syb6330), and 25 μg of total protein lysate from mixed stage population were loaded per lane and specific proteins were detected with an anti-FLAG antibody and enhanced chemiluminescence. Protein loading was analyzed by No-stain protein labeling reagent. (E) Densitometric quantification of MRPL-58 protein levels in western blots. Data are expressed as mean ± SEM from 3 biological replicates. Asterisks indicate statistically significant difference relative to wild type, Student’s t test (*p < 0.05). Underlying data can be found in S1 Data.