Fig 1: Increased DNA damage and impaired DDR in Hnrnprtm1a/tm1a motoneurons. (A) Western blot analysis of subcellular fractions from control motoneurons (0 Gy) or motoneurons subjected to ?-irradiation (9 Gy). Motoneurons were fractioned into cytosolic (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions. Fractions were probed with the indicated antibodies. (B) Western blot analysis of chromatin fractions of NSC-34 cells incubated with Benzonase (Bn) or the indicated amounts of RNase A and separated into supernatant and pellet by centrifugation. (C) Immunoprecipitation of ?-H2AX from nuclear fractions of control (0 Gy) or irradiated (9 Gy) NSC-34 cells. Proteins were analyzed by western blot using antibodies against ?-H2AX or hnRNP R. In, input; IP, immunoprecipitation. (D) Representative images of alkaline comet assays performed on Hnrnpr+/+ and Hnrnprtm1a/tm1a motoneurons cultured for 6 DIV under control conditions (0 Gy) and after exposure to ?-irradiation (9 Gy). (E) Quantification of comet mean tail moments. Data are mean ± SD (n = 3 independent experiments; N = 71 nuclei for Hnrnpr+/+ (0 Gy), N = 55 nuclei for Hnrnpr+/+ (9 Gy), N = 82 nuclei for Hnrnprtm1a/tm1a (0 Gy) and N = 76 nuclei for Hnrnprtm1a/tm1a (9 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P = 0.01. (F) Representative images of ?-H2AX immunofluorescence staining of Hnrnpr+/+ and Hnrnprtm1a/tm1a motoneurons under non-irradiated conditions (0 Gy) or after exposure to ?-irradiation (9 Gy) followed by the indicated recovery time. Scale bar: 5 µm. (G) Quantification of nuclear ?-H2AX immunostaining shown in (F). Data are mean ± SD (n = 4 independent experiments; N = 38 nuclei for Hnrnpr+/+ (0 Gy), N = 40 nuclei for Hnrnpr+/+ (9 Gy, 3 min recovery), N = 40 nuclei for Hnrnpr+/+ (9 Gy, 10 min recovery), N = 41 nuclei for Hnrnpr+/+ (9 Gy, 30 min recovery), N = 40 nuclei for Hnrnpr+/+ (9 Gy, 24 h recovery), N = 42 nuclei for Hnrnprtm1a/tm1a (0 Gy), N = 39 nuclei for Hnrnprtm1a/tm1a (9 Gy, 3 min recovery), N = 41 nuclei for Hnrnprtm1a/tm1a (9 Gy, 10 min recovery), N = 40 nuclei for Hnrnprtm1a/tm1a (9 Gy, 30 min recovery) and N = 39 nuclei for Hnrnprtm1a/tm1a (9 Gy, 24 h recovery)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P = 0.01, ***P = 0.001. (H) Western blot analysis of hnRNP R levels in cultured motoneurons transduced with control or shRNA against both hnRNP R isoforms (shRNA-FL+?N). Gapdh was used as loading control. (I) Representative images of alkaline comet assay performed on motoneurons transduced with control and shRNA-FL+?N under non-irradiated conditions (0 Gy) or after exposure to ?-irradiation (9 Gy). (J) Quantification of comet mean tail moments. Data are mean ± SD (n = 3 independent experiments; N = 32 nuclei for Control (0 Gy), N = 29 nuclei for Control (9 Gy), N = 37 nuclei for shRNA-FL+?N (0 Gy) and N = 30 nuclei for shRNA-FL+?N (0 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P = 0.01. (K) Representative images of ?-H2AX immunofluorescence staining of control and shRNA-FL+?N-transduced motoneurons under non-irradiated conditions (0 Gy) or after exposure to ?-irradiation (9 Gy) followed by the indicated recovery time. EGFP was used as a marker to identify transduced cells. Scale bar: 5 µm. (L) Quantification of nuclear ?-H2AX immunostaining in (K). Data are mean ± SD (n = 3 independent experiments; N = 34 nuclei for control (0 Gy), N = 32 nuclei for control (9 Gy, 10 min recovery), N = 22 nuclei for control (9 Gy, 24 h recovery), N = 21 nuclei for shRNA-FL+?N (0 Gy), N = 33 nuclei for shRNA-FL+?N (9 Gy, 10 min recovery), N = 36 nuclei for shRNA-FL+?N (9 Gy, 24 h recovery)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; ***P = 0.001. (M) Representative images of ?-H2AX immunofluorescence staining of motoneurons transduced with control, shRNA-FL+?N, shRNA-FL+?N; ?N(OE) or shRNA-FL+?N; FL(OE) constructs under non-irradiated conditions (0 Gy) or after exposure to ?-irradiation (9 Gy) followed by 10 min recovery. EGFP was used as a marker to identify cells transduced with the control or shRNA-FL+?N construct. The rescue constructs shRNA-FL+?N; ?N(OE) and shRNA-FL+?N; FL(OE) expressed EGFP-tagged hnRNP R-?N or hnRNP R-FL, respectively, in addition to an shRNA targeting both endogenous hnRNP R isoforms. Scale bar: 5 µm. (N) Quantification of nuclear ?-H2AX immunostaining in (M). Data are mean ± SD (n = 3 independent experiments; N = 25 nuclei for control (0 Gy), N = 25 nuclei for control (9 Gy), N = 25 nuclei for shRNA-FL+?N (0 Gy), N = 25 nuclei for shRNA-FL+?N (9 Gy), N = 25 nuclei for shRNA-FL+?N; ?N(OE) (0 Gy), N = 25 nuclei for shRNA-FL+?N; ?N(OE) (9 Gy), N = 25 nuclei for shRNA-FL+?N; FL(OE) (0 Gy) and N = 25 nuclei for shRNA-FL+?N; FL(OE) (9 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; ***P = 0.001.
Fig 2: Direct interaction of hnRNP R and SMN.(A) Purification scheme of recombinant hnRNP R and SMN expressed as His-tagged proteins in E. coli strain BL21. (B) Affinity purification profile on a fast protein liquid chromatography (FPLC) of hnRNP R and SDS-PAGE of recombinant hnRNP R purification steps visualized by silver staining. (C) Affinity purification profile on a FPLC of SMN and SDS-PAGE of recombinant SMN purification steps visualized by colloidal staining. (D) Coimmunoprecipitation of recombinant SMN and hnRNP R.
Fig 3: Subcellular distribution of Smn and hnRNP R in isolated embryonic motoneurons.(A) Motoneurons showed reduced Smn protein levels upon lentiviral knockdown of Smn. Uninfected or GFP-infected mouse embryonic motoneurons were used as controls. Levels of calnexin and hnRNP R were not affected. For this experiment a C-terminal antibody directed against hnRNP R was used as reported recently [29]. This antibody recognizes distinct hnRNP R isoforms. (B) Representative images of motoneurons cultured for 7DIV and labeled against Smn (scale bar: 10 µm). GFP-transfected controls revealed immunoreactive signals for Smn in the cytosol, in neuronal processes and in Gem-like nuclear structures. Upon lentiviral Smn knockdown both cytosolic Smn immunoreactivity (Uninfected set as ‘1’, n = 4, N = 51; GFP 1.02±0.04, n = 4, N = 60; sh-Smn 0.34±0.02, n = 4, N = 74; P<0.001, t = 19.19) and number of Gems per nucleus (Uninfected 1.03±0.18, n = 4, N = 51; GFP 0.97±0.15, n = 4, N = 60; sh-Smn 0.08±0.02, n = 4, N = 74; P<0.01, t = 4.929) were significantly reduced in comparison to uninfected cells. (C) Subcellular distribution of hnRNP R in soma, axon and growth cone of primary motoneurons cultured for 5DIV and costained against synaptophysin (SynPhys) and neurofilament (NF-L) (scale bar: 10 µm (upper row), 5 µm). (D) Lentiviral knockdown of hnRNP R led to a dose-dependent reduction of hnRNP R levels. Calnexin and Smn protein were not altered significantly. (E) HnRNP R knockdown was also detected by immunofluorescence validating the used antiserum peptide ICN 1-18 (GFP 1.00±0.04, n = 8, N = 100; sh-hnRNP R 0.48±0.04, n = 6, N = 63; P<0.0001, t = 8.719, DF = 12) (scale bar: 10 µm).
Fig 4: Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm.Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ?-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53], [56]. The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ?-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).
Fig 5: Recruitment of Yb1 to chromatin in response to DNA damage is impaired in the absence of the full-length hnRNP R isoform. (A) Non-irradiated NSC-34 cells (0 Gy) or NSC-34 cells subjected to ?-irradiation (9 Gy) were separated into cytosolic (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions. Fractions were analyzed by western blot with the indicated antibodies. (B) Quantification of western blot data shown in (A). The expression of Yb1 in each fraction is presented as the percentage of total. Data are mean ± SD (n = 3 independent experiments). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; *P = 0.05. (C) Non-irradiated (0 Gy) or irradiated (9 Gy) NSC-34 cells transduced with control lentivirus or lentivirus expressing shRNA-FL for knockdown of full-length hnRNP R were fractionated and analyzed by western blot with the indicated antibodies. (D, E) Quantification of the Yb1 Western blot data shown in (C) for control conditions (0 Gy) (D) and after exposure to ?-irradiation (9 Gy) (E). The expression of Yb1 in each fraction is presented as the percentage of total. Data are mean ± SD (n = 3 independent experiments). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P = 0.01, ***P = 0.001. (F) Immunoprecipitation of ?-H2AX from lysates of non-irradiated (0 Gy) or irradiated (9 Gy) NSC-34 cells transduced with control lentivirus or lentivirus expressing shRNA-FL for knockdown of full-length hnRNP R. Proteins were analyzed by Western blot with the indicated antibodies. In, input; IP, immunoprecipitation.
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