Fig 2: RBM45 interacts with TDP-43 and FUS.(a) FLAG-RBM45 was expressed in HEK293 cells and immunoprecipitations with FLAG antibody or IgG were performed. Live cells were treated with 0.1% formaldehyde to cross-link proteins prior to cell lysis and immunoprecipitation. The crosslinking was reversed by heating in SDS-sample buffer prior to SDS-PAGE. The IP fractions were immunoblotted with TDP-43, FUS, FLAG (FLAG-RBM45) and GAPDH (negative control) antibodies. (b) HA-RBM45 was transfected into HEK293 cells stably expressing FLAG-TDP-43 (left) or FLAG-FUS (right). Immunoprecipitations and immunoblot were performed as described in a. HA-RBM45 was detected with HA antibody. (c) In-cell RNase treatment and crosslinking-IP were performed on cells expressing FLAG-RBM45. The amount of TDP-43, but not FUS, that co-purified with FLAG-RBM45 was reduced with the RNase treatment. (d) In-cell FLAG-RBM45 and TDP-43 interactions as demonstrated by Proximity Ligation Assay (PLA) in the nucleus of HEK293 cells. PLA was performed on HEK293 cells stably expressing FLAG-RBM45. Each red dot indicates the protein-protein interaction (<40 nm) between FLAG-RBM45 and endogenous TDP-43. The primary antibodies are listed on top of each panel. DAPI was used as the nuclear stain. Scale bar: 10 μM. (e) Wild-type and cytoplasmic retained RBM45 have similar binding efficiency with TDP-43. HA-tagged RBM45 wild-type or the NLS M2/3 mutant was transfected into the HEK293 cell line stably expressing FLAG-TDP-43. FLAG-TDP-43 immunoprecipitation and immunoblot were performed as described in b. (f) PLA assays showing the in-cell protein-protein interactions between HA-RBM45 wild-type or NLS M2/3 mutant and endogenous TDP-43 in HEK293 cells. HA-RBM45 constructs were expressed in HEK293 cells and PLA assays were performed. DAPI was used as the nuclear stain. Scale bar: 10 μM. (g) The self-association deficient RBM45 exhibits reduced binding to TDP-43. HA-tagged full-length, D1 and D4 RBM45 constructs were transfected into the HEK293 cell line stably expressing FLAG-TDP-43. FLAG-TDP-43 immunoprecipitation and immunoblot were processed as in b. (h) The self-association deficient RBM45 exhibits reduced binding to FUS. HA-tagged full-length, D1 and D4 RBM45 constructs were transfected into the HEK293 cell line stably expressing FLAG-FUS. FLAG-FUS immunoprecipitation and immunoblot were processed as described in b.

Fig 3: RBM45 homo-oligomerization mediates its incorporation into stress granules and TDP-43 association.(a) The cytoplasmic granules containing the RBM45 HA-NLS mutant are immunoreactive with stress granule markers TIAR (top) as well as TDP-43 (middle), but are not immunoreactive with FUS (bottom). HA-RBM45 NLS M2 mutant (from Fig. 1) was transfected into SK-N-SH cells, and 48 hr post-transfection, the transfected cells were stressed with 1 mM sodium arsenite for 30 minutes followed by immunostaining. HA-RBM45 NLS was immunostained with rabbit anti-HA antibody (green). Nuclei were labeled with DAPI. Scale bar: 10 μm. TIAR was stained with mouse monoclonal anti-TIAR antibody, TDP-43 was stained with mouse monoclonal anti-TDP-43 antibody that recognizes full-length as well as the cleaved C-terminal fragment of TDP-43, and FUS was stained with mouse monoclonal anti-FUS antibody. (b) RBM45-NLS mutant with additional HOA domain deletion (NLS/D5 construct) are not incorporated into cytoplasmic granules and fail to co-localize with TIAR (top) or TDP-43 (bottom). Transfection and immunofluorescence experiments were performed as described in a. (c,d) Quantification demonstrating the percentage of the cells transfected with NLS or NLS/D5 constructs that form cytoplasmic granules positive for TIAR in c or TDP-43 in d (n = 3; cell numbers > 50 per experiment). The p-value was calculated by two-tailed paired t-test. Error bar represents SEM.

Fig 4: RBM45 forms high-molecular-weight oligomers.(a) Diagram of the DSS crosslinking experiments. Live HEK293 cells constitutively expressing FLAG-RBM45 underwent in vivo crosslinking using the cell-permeable NHS-ester crosslinker DSS with increasing concentrations from 0 to 5000 μM. The cells were lysed and lysates were run on denaturing SDS-PAGE and probed for immunoblot. (b) Immunoblot analysis of samples generated from a. Samples probed with RBM45 C-terminal antibody showed that FLAG-RBM45 in non-crosslinked cells migrates at the expected molecular weight of a monomer (56 kD), whereas FLAG-RBM45 in DSS-treated cells migrates at the molecular weight of a dimer (~110 kD) or a pentamer (~280 kD). The arrows indicate the sizes of FLAG-RBM45 monomer and oligomers. Samples probed with TDP-43 antibody revealed the TDP-43 monomer (45 kD) and TDP-43 dimer (~90 kD). Samples probed with β-tubulin and GAPDH antibodies were negative control to rule out non-specific crosslinking of cellular monomeric proteins. (c) HA-tagged full-length RBM45 and RRM1-deletion construct (107–474) were expressed in HEK293 cells and DSS in vivo crosslinking (1 mM DSS for 10 minutes) were performed. The oligomerization of the HA-RBM45 constructs was examined by immunoblot and probed with RBM45 C-terminal antibody. Filled arrowheads indicate the monomers, while open arrowheads indicate the pentamers. The observed molecular weights of the monomers and pentamers are indicated next to the bands. The theoretical molecular weights of the monomers and pentamers are indicated below the blot. (d) The HA-tagged D1-D5 constructs (Fig. 2f) were tested for their ability to oligomerize as described in c. Filled arrowheads indicate the monomers, while open arrowheads indicate the pentamers. The theoretical molecular weights of the monomers and pentamers are indicated below the blot. The blot was also probed with TDP-43 (crosslinking positive control) and GAPDH (negative control) antibodies. Full length gels are shown in Supplemental Figure S2b. (e) RBM45 homo-oligomerization occurs in both the nucleus and cytoplasm. The oligomerization of HA-tagged RBM45 or the NLS M2/3 mutant (from Fig. 1) was examined by immunoblot and probed with HA antibody. Arrowheads denote HA-RBM45 and oligomer species. (f) Subcellular fractionation of the HA-RBM45 constructs in e. Fractionation was performed as described in Fig. 1d.