Abnormally increased interaction of ^muHspB8 with Ddx20 in cell extracts. HEK293T cells were singly or doubly transfected with vector cDNA of Xpress-tagged Ddx20 (construct 5) and myc-tagged ^wtHspB8 (construct 6), ^K141EHspB8 (construct 7), or ^K141NHspB8 (construct 8), as indicated. Forty-eight hours later, the presence of the expressed proteins in cell extracts was confirmed by SDS-PAGE/Western blotting using Xpress- and myc-specific antibodies (I, II). ^wtHspB8 and ^muHspB8 occur in similar amounts in the cell extracts. Subsequently, Ddx20 was immunoprecipitated from the cell extracts using both low and high stringency conditions (III and V, respectively), and its presence in the precipitates was confirmed in cells transfected with Ddx20 indicating proper functioning of the method. The Western blots of the precipitates were then developed for co-precipitated HspB8. The various HspB8 forms were detected only in the precipitates of those cells which expressed both proteins, both under low and high stringency conditions (IV and VI, respectively). This co-IP indicates interaction between Ddx20 and the various forms of HspB8. At low stringency conditions (IV), all HspB8 forms were co-precipitated to a similar extent, while at high stringency conditions (VI), the amounts of co-precipitated ^K141NHspB8, and to a lesser extent also of ^K141EHspB8, were greater than that of ^wtHspB8. The blot is representative of three independent experiments

In vivo interaction of ^wtHspB8 and ^muHspB8 species with Ddx20. a HEK293T cells grown on coverslips were doubly transfected with vector cDNA of Xpress-tagged Ddx20 (construct 5) and FLAG-tagged ^wtHspB8 (construct 9), ^K141EHspB8 (construct 10), or ^K141NHspB8 (construct 11), as indicated. Twenty-four hours later, cells were fixed and processed for immunostaining. In the selected cells, expressed Ddx20 (red) and all HspB8 species (green) showed a relatively even distribution in the cytoplasm (co-localization in yellow). For comparison, nuclei of the same cells stained with DAPI are shown. The bar indicates 50 μM. b HEK293T cells grown on glass bottom culture plates were doubly transfected with vector cDNA of CIT-tagged Ddx20 (construct 12) and CFP-tagged ^wtHspB8 (construct 13), ^K141EHspB8 (construct 14), or ^K141NHspB8 (construct 15), or with control vectors, as indicated. Ten hours later, in vivo images of selected cells with relatively even cytoplasmic distribution of the expressed proteins were collected. Such cells were used for the qFRET analysis as shown in (d). c SDS-PAGE/Western blotting of the cells as shown in (b). For visualization of the HspB8-CFP species, an anti-GFP antibody was used. For visualization of the Ddx20-CIT, an anti-Ddx20 antibody was used which detected also the endogenous Ddx20 (control lane). Note that the transfected cells contained similar amounts of ^wtHspB8-CFP, ^K141EHspB8-CFP, and ^K141NHspB8-CFP (left panel), and also similar amounts of Ddx20-CIT (right panel). The positions of molecular mass marker proteins are indicated. d AAFE values for the interactions of Ddx20 with ^wtHspB8, ^K141EHspB8, and ^K141NHspB8 as determined by the qFRET method. The used cells and constructs were as in (b). One-way ANOVA analysis revealed significant differences between the four groups, control 1, ^wtHspB8, ^K141EHspB8, and ^K141NHspB8 [F(5, 234) = 269.25; P < 0.001]. Post-hoc pairwise group comparisons showed significant differences between the sample values and the control (P < 0.001), between ^K141NHspB8 and ^wtHspB8 (P < 0.001), and between ^K141NHspB8 and ^K141EHspB8 (P < 0.001). By this method, no significant difference between ^K141EHspB8 and ^wtHspB8 (P = 0.099) was revealed, while the direct comparison between both sample groups by the Student’s t test indicated a significant difference (P = 0.033)

Schematic of protein interactions involving Ddx20 and HspB8. a Identified proteins that interact with Ddx20 and the cellular processes in which they are involved. Upon binding to Ddx20, HspB8 theoretically may modulate these cellular processes. Gemin4 and SMN protein are components of the SMN complexes that are involved in spliceosome assembly and RNP processing. Upon binding to gemin4 and eIF2C2, Ddx20 forms complexes with microRNAs that are involved in gene silencing and translational repression, and upon binding to Ago2, Ddx20 may modulate siRNA biogenesis. Ddx20 can also interact with the nuclear proteins and transcription factors that are involved in transcriptional repression, including Epstein–Barr virus nuclear proteins EBNA2 and 3C, nuclear receptor steroidogenic factor SF-1, transcription factor FOXL2, early growth response transcription factors Egr1-4, and the mitogenic Ets repressor METS with its co-repressor components N-coR and Sin3A, and with the histone-deacetylases HDAC 2 and 5. b Through protein interactions, four etiologic factors HspB1, HspB8, HspB5, and SMN protein have been linked that are associated with the various forms of motor neuropathy (dHMN, CMT, SMA) or myopathy. Based on the identified interactions, Ddx20 is a candidate gene for mutations in neuromuscular or muscular diseases of unknown cause. While sHSP complexes may contain various family members, the recruitment of HspB8 into true hetero-oligomeric complexes, together with HspB1 and HspB5, remains to be determined. SMN complexes contain a number of additional core components and associated proteins (not shown). The HspB8–Ddx20 interaction apparently involves RNA. c Changed conformation of ^muHspB8 leads to increased interactions with HspB1 and HspB5 (Fontaine et al. 2006), and with Ddx20. At the same time, the associated RNA becomes exposed to RNase

Sequences and domains of human HspB8 and Ddx20 and isoelectric points of ^wtHspB8 and ^muHspB8 forms. a HspB8 (GenBank accession: AF250138.1). The domains were designated as described previously (Fontaine et al. 2003). The known disease-associated mutations affect Lys141 (highlighted in black) in the conserved α-crystallin domain. b Ddx20 (GenBank accession: NM_007204.4). The N-terminal half contains the conserved DEAD box RNA helicase domain with the seven helicase motifs I, Ia, II, III, IV, V, and VI. The non-conserved C-terminal half contains the HspB8 binding region (positions 475–727; bold, single underline), the SMN protein-binding region (positions 456–547; dashed underline; cf. Charroux et al. 1999), and the SF-1 binding region (positions 719–824; dotted underline; corresponds to positions 721–825 in the mouse sequence BC137721.1; cf. Yan et al. 2003). In (a) and (b), cysteine residues reactive to the BMH cross-linking reagent are highlighted in gray (cf. Fig. 3). c Isoelectric focusing of ^wtHspB8 and ^muHspB8 forms. cDNAs of untagged ^wtHspB8 (construct 2), ^K141EHspB8 (construct 3), or ^K141NHspB8 (construct 4) were used to transfect HEK293T cells, and 48 h later the cell proteins were separated by IEF-PAGE/Western blotting using a specific anti-HspB8 antibody. ^K141EHspB8 and ^K141NHspB8 showed more acidic pIs (∼4.2 and ∼4.3, respectively) as compared to ^wtHspB8 (∼4.7). The approximate positions of the marker proteins β-lactoglobulin B (∼5.1), phycocyanin (∼4.6), and glucose oxidase (∼4.2) are indicated

Formation of heterogeneous high-molecular-mass complexes containing Ddx20 and HspB8 species. a HEK293T cells were singly or doubly transfected with vector cDNA of Xpress-tagged Ddx20 and myc-tagged HspB8 species as indicated using the same constructs as in Fig. 2. Forty-eight hours later, cells were treated for 30 min with the cross-linker BMH, and the cross-linked proteins were analyzed by SDS-PAGE/Western blotting using an Xpress-specific antibody to detect Ddx20. Only in the presence of either ^wtHspB8 or ^muHspB8, Ddx20 was cross-linked into high-molecular mass material (bands 1–3, band region 4 in lanes 6–8). On the same blots, actin was visualized to demonstrate equal loading of protein onto gels. b The experiment was performed as in (a) using a myc-specific antibody to detect the various HspB8 forms on the Western blots. Only in the presence of Ddx20, were ^wtHspB8 or ^muHspB8 cross-linked into high-molecular mass material (bands 2, 3 in lanes 10–12), in addition to the formation of HspB8 dimers (upper panel). Analysis of the same samples revealed that actin was not cross-linked into any high-molecular weight material suggesting that cross-linking of the HspB8 species with Ddx20 resulted from a specific interaction, and not from a general, non-specific cross-linking of cell proteins (lower panel). c The same sample as shown in lanes 6 and 10 in (a) and (b), respectively, was loaded onto two adjacent lanes of a SDS gel. One lane of the Western blot was developed for Ddx20, the other for ^wtHspB8. The identical position of bands 2 and 3 suggests that this cross-linked material contains both Ddx20 and ^wtHspB8. The positions of molecular mass markers (kDa), Ddx20 monomers, and of HspB8 monomers and dimers are indicated in a–c. The results are representative of three independent experiments

Involvement of RNA in the Ddx20–HspB8 interaction. HEK293T cells were singly or doubly transfected with vector cDNA of Xpress-tagged Ddx20 (construct 5) and myc-tagged ^wtHspB8 (construct 6), ^K141EHspB8 (construct 7), or ^K141NHspB8 (construct 8), as indicated. Forty-eight hours later, the presence of the expressed proteins in cell extracts was confirmed by SDS-PAGE/Western blotting using Xpress- and myc-specific antibodies (I, II). Immunoprecipitation of the various HspB8 species after RNase treatment vs. the controls without treatment resulted in similar amounts of precipitated proteins (III). The amounts of Ddx20 co-immunoprecipitated with ^wtHspB8 with and without RNase treatment were similar (IV, lanes 3 and 4), while the amounts of Ddx20 co-immunoprecipitated with ^K141EHspB8 and ^K141NHspB8 were decreased after RNase treatment as compared to the untreated samples (IV, lanes 5–8). The blot is representative of three independent experiments