Fig 1: RBM45 subcellular distribution and inclusion pathology in FTLD-TDP, ALS, and AD. Immunohistochemistry for RBM45, SAFB, and TDP-43 was performed in hippocampal dentate gyrus from non-neurologic disease control, FTLD-TDP, ALS, and AD subjects. a Control subjects showed strong immunoreactivity for SAFB, TDP-43, and RBM45, with RBM45 immunoreactivity concentrated in the perinucleolar region (arrowheads) and SAFB and TDP-43 diffusely localized throughout the nucleus. b RBM45 immunoreactivity was predominantly in nuclear inclusions in FTLD dentate gryus cells, with attendant loss of cytoplasmic and perinucleolar immunoreactivity. Nuclear RBM45 inclusions lack SAFB and TDP-43. Dentate gyrus cells containing RBM45 nuclear inclusions also exhibit reduced SAFB immunoreactivity. Several TDP-43 and RBM45-positive cytoplasmic inclusions were found in FTLD subjects (arrow). c As in (b), but for ALS subjects. Nuclear RBM45 inclusions lack SAFB or TDP-43, and cytoplasmic inclusions contain RBM45 and TDP-43 (arrow). d As in (b), but for AD subjects. Nuclear RBM45 inclusions lack SAFB and TDP-43, and cytoplasmic inclusions contain RBM45 and TDP-43 (arrow). e Boxplot showing the number of RBM45 nuclear inclusions by group (# = p < 0.01, * = p < 1 × 10− 6). f Stacked bar plot showing the number of RBM45 nuclear inclusions per cell by subject group. Most dentate gyrus cells from control subjects have no inclusions, while FTLD-TDP, ALS, and AD subjects have significantly more granules per cell across the range of observed values. g The relationship between RBM45 nuclear inclusions and SAFB immunoreactivity is shown, along with the regression line. Regardless of disease state, with increasing numbers of RBM45 nuclear inclusions, SAFB immunoreactivity decreases, particularly at ≥3 granules per cell. The relationship between these two variables is statistically significant (p < 1 × 10− 16). In the scatterplot shown, jitter is added to the points to more clearly visualize the data, but the separation between inclusion integer numbers is still seen. The inset image shows an example of adjacent cells in an FTLD-TDP subject where one cell with no RBM45 granules has strong SAFB nuclear immunoreactivity, while the neighboring cell has several nuclear RBM45 inclusions and low SAFB nuclear immunoreactivity. In (a-d), the scale bar = 20 μm, while in the inset in (g) the scale bar = 5. For all panels, FTLD = FTLD-TDP
Fig 2: Domains required for RBM45 incorporation into nuclear stress bodies (NSBs). a Schematic showing functional domains and their position in the full-length RBM45 protein. RRM = RNA recognition motif, HOA = homo-oligomer assembly domain, NLS = nuclear localization sequence. HEK293 cells were transfected with constructs encoding N-terminally HA-tagged wild-type (WT) or domain-modified forms of RBM45 as indicated to determine which domains of the protein are necessary for incorporation into NSBs. b In unstressed cells, the distribution of WT RBM45 is diffuse and nuclear and no NSBs are observed. c Heat shock (42 °C for 2 h) leads to the robust formation of NSBs that are positive for wild-type (WT) RBM45 and the NSB marker HSF1. d Removal of the RBM45 NLS (∆NLS) leads to its sequestration in the cytoplasm and prevents its association with NSBs during heat shock. e Removal of RRM1 (∆RRM1) does not alter the association of RBM45 with NSBs. f Removal of RRM2 (∆RRM2) prevents the association of RBM45 with NSBs. g Removal of RRM3 (∆RRM3) prevents the association of RBM45 with NSBs. h Removal of the RBM45 HOA domain (∆HOA) does not alter the association of RBM45 with NSBs. For all images, scale bar = 5 μm
Fig 3: Live-cell imaging of RBM45 nuclear stress body (NSB) and nuclear inclusion formation during conditions of cellular stress. HEK293 cells induced to express N-terminal GFP-tagged RBM45 were used to visualize the subcellular distribution of RBM45 during normal conditions and during acute and chronic cellular stress. a In untreated cells, RBM45 is predominantly nuclear and diffusely localized throughout the nucleus. b Treatment with 0.5 mM sodium arsenite for 1 h causes the formation of numerous RBM45-positive nuclear stress bodies (NSBs; arrowheads). Cells were treated with 0.5 mM sodium arsenite at t = 0 h. Under conditions of acute cellular stress, NSB formation is reversible, most RBM45-positive NSBs have dissipated by t = 6 h, and RBM45 remains diffusely localized throughout the nucleus for the remainder of the experiment. c Chronic cellular stress leads to the entrapment of RBM45 in nuclear inclusions. Cells were treated with 0.1 mM sodium arsenite for 12 h to assess the effect of chronic cellular stress on RBM45 subcellular distribution. Because of the lower concentration of sodium arsenite, the time course of NSB formation is altered. Following removal of sodium arsenite (panel “Washout”), RBM45 remains in nuclear inclusions for the remaining 12 h duration of the experiment (arrowheads). For all panels, scale bar = 5 μm
Fig 4: Schematic overview of RBM45 domains structure and protein-protein interactions.(a) Domain structure of RBM45 with the newly identified HOA (homo-oligomer assembly) domain highlighted in blue and the NLS in red. (b) A working model for RBM45 homo-oligomerization and distribution in the cell. RBM45 monomers assemble into a ring-shape pentamer which may dimerize to form a decamer. The RBM45 homo-oligomer acts as a scaffold to associate with TDP-43 mediated by RNA in the nucleus or cytoplasm. Accumulation of RBM45 in the cytoplasm leads to stress granule inclusion together with TIAR and TDP-43.
Fig 5: Nuclear localization of RBM45.(a) Immunofluorescence of endogenous RBM45 in SHSY5Y and Neuro2A cells that were stained with RBM45 C-terminal antibody (epitope residues 460–474). DAPI was used as the nuclear stain. Scale bar: 10 μm. (b) Overexpressed EGFP-RBM45 forms large cytoplasmic granules in Neuro2A cells. Images were taken with living cells grown on glass-bottom plates. Phase contrast is used to delineate cell shape. The cytoplasmic granules are indicated with white arrows. Scale bar: 10 μm. (c) Domain structure of RBM45. The RRM domains are shown in yellow. The two predicted NLS are shown as dark grey boxes with their sequences indicated below (residues 376–385, and residues 454–472). The basic residues are highlighted in blue. The NLS mutants M1 and M2 with mutated amino acid residues highlighted in red are shown below the wild-type sequences. (d) Subcellular fractionation and immunoblot of the HA-tagged wild-type, M1 mutant, M2 mutant and the double-mutant M1/2 of RBM45. HA-RBM45 constructs were transfected into Neuro2A cells and biochemical fractionation was performed 48 hr post-transfection. Equal proportions of nuclear extract and cytoplasmic extract were immunoblotted with HA (HA-RBM45 constructs), GAPDH (cytoplasmic marker) and Lamin A/C (nuclear marker) antibodies. C = Cytoplasmic fraction, N = Nuclear fraction. The average percentage of nuclear RBM45 in total RBM45 from three experiments and standard deviation are indicated below the blot. The p-value was calculated by two-tailed paired t-test. (e) Immunofluorescence showing the subcellular localization of the HA-RBM45 wild-type and the M2 mutant in Neuro2A cells. Ectopic HA-RBM45 was stained with mouse HA antibody (red). Total RBM45 was stained with RBM45 C-terminal antibody (green). Nuclei were stained with DAPI. Scale bar: 10 μm. (f) Sequence of the bipartite NLS and its mutants. The basic residues are shown in blue and the mutated residues are highlighted in red. M2 mutant contains mutations at the C-end, M3 mutant contains mutations at the N-end, and the double mutant M2/3 contains both mutations at each end. (g) Subcellular fractionation and immunoblot of HA-tagged RBM45 wild-type and NLS mutants from f. Transfection, fractionation, immunoblot and statistics were performed as described in d.
Supplier Page from DNASU for RBM45 (Homo sapiens) in pANT7_cGST (GST-tagged in vitro expression vector)