Fig 1: ATAD5 interacts with DEAD-box RNA helicases and DHX9. (A) HEK293T cells were transfected with strep-Tag II-ATAD5 cDNA. After 48 h, proteins were extracted and subjected to affinity purification-mass spectrometry analysis. The numbers of peptide hits for some selected proteins from the analysis were displayed. Proteins in red indicate DEAD-box RNA helicases and DHX9. (B–E, K) After transfection of HEK293T cells as indicated, nuclear extracts were prepared for immunoprecipitation (IP) with an anti-FLAG antibody. Immunoprecipitates were eluted, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (F–I) Nuclear extracts from HEK293T cells were immunoprecipitated with anti-ATAD5 antibody (F, G) or anti-DDX5 antibody (H, I). (H, I) Cells were transfected with UAF1. (G, I) Relative band intensities were quantified and plotted, N=4. Statistical analysis: two-tailed paired Student's t-test; **** P < 0.001, **P < 0.01, *P < 0.05, ns, not significant. (J) Purified MYC-FLAG-DDX5, His-UAF1, and His-FLAG-ATAD5-N400 (ATAD5 1–400 aa) proteins were incubated as indicated and pulled down with an anti-MYC antibody. Purified FLAG-His-MBP-TALE protein was included as a negative control. (K) HEK293T cells were transfected with FLAG-tagged DDX5 and one of S-tagged UAF1 deletion mutants (S-UAF1WD40 and S-UAF1SLD). (L) HeLa-TetOn-RNaseH1-GFP cells were transfected with UAF1 siRNA, treated with doxycycline to express RNaseH1 for 48 h and immunostained with the S9.6 antibody. The intensity of S9.6 staining was quantified and plotted. Three independent experiments were performed, and one representative result was displayed. Red bar indicates mean value. Statistical analysis: two-tailed Student's t-test; * P < 0.05 and ns, not significant.
Fig 2: ATAD5 depletion reduces the association of RNA helicases with normal and stalled replication forks. (A) HEK293T cells were labeled with EdU for 20 min and then treated with 10 μM thymidine for indicated times. Whole cell extracts were prepared for an iPOND EdU-thymidine chase assay. (B) HEK293T cells were transfected with ATAD5 siRNA for 48 h then whole cell extracts were prepared for an iPOND assay. (C–F) U2OS-TetOn-ATAD5 cell lines were transfected with ATAD5 siRNAs and treated with doxycycline to express wild type ATAD5 (ATAD5WT) or UAF1 interaction defective mutant ATAD5 (ATAD5ΔUAF1). After 48 h, cells were fixed for a SIRF assay on DDX21 (C, D) or DDX5 (E, F). Hydroxyurea (HU) was added at a final concentration of 2 mM for 3 h before fixation. (C, E) Representative SIRF images. (D, F) Number of nuclear SIRF signal foci were counted and plotted. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed Student's t-test; **** P < 0.001, *** P < 0.005, **, P < 0.01, * P < 0.05 and ns, not significant.
Fig 3: DDX5,17 affect the splicing of developmentally-regulated and MBNL-dependent AS events in muscle cells. A Mbnl1, Mbnl2, Ddx5 and Ddx17 gene expression during development of skeletal muscles (n = 1; [16]) and the brain (n = 2; [65]) determined by RNA-Seq expression analyses and using DESeq2-normalization method. E and PN correspond to embryonic and postnatal stages of development, respectively. B Mbnl1, Mbnl2, Ddx5 and Ddx17 gene expression across five stages of murine muscle development (E18.5, PN1, PN5, PN14, PN90) determined by RT-qPCR. Data represent the mean ± SD (n = 9). Statistical significance was calculated in reference to embryonic day 18.5 (E18.5) using Student’s t test; ns nonsignificant, ∗∗∗ for P < 0.001. C Representative western blot analyses of Mbnl1, Mbnl2, Ddx5 and Ddx17 protein levels in four murine muscle developmental stages (E19, PN1, PN5, adult ) (n = 2). Vinculin serves as a loading control. D Representative western blot analyses of Mbnl1, Mbnl2, Ddx5 and Ddx17 protein levels in adult murine skeletal muscles, heart and brain (n = 3). Vinculin serves as a loading control. E Genome browser view (mm10) of RNA-seq AS analyses shows MBNL-dependent AS events, Atp2a1 e22 and Nfix e7, whose splicing changes during muscle development. F Representative western blot analyses of protein levels in HSkM upon siDDX5,17 treatment. VINCULIN serves as loading controls. Each loading control corresponds to the proteins above it. The image presents juxtaposed lanes that were non-adjacent in the blot. The lanes separation is delineated by a black separation. Blot images of all samples and calculated protein levels are shown in Suppl. Figure S2C. All samples derive from the same experiment and the blots were processed in parallel. G The pie chart summarizes the RT-PCR screening for AS events shared by MBNL1 and DDX5,17 in siDDX5,17- and siMBNL1-treated HSkM. The AS events were categorized into a group of shared events if PSI ≥ 5% and P < 0.05 in Student’s t test. AS analyses of each event are presented in Suppl. Figure S2B. H Representative gels and calculations of RT-PCR analyses of shared AS events in siRNA treated HSkM (n = 3), human adult muscles (n = 3) and across five stages of murine muscle development (n = 4). Data represent the mean PSI values ± SD. Statistical significance was calculated in reference to control (siCtrl) or E18.5 using Student’s t test; ns nonsignificant, ∗ for P < 0.05, ∗∗ for P < 0.01, ∗∗∗ for P < 0.001. I The pie chart shows the number of shared AS events analyzed in HSkM, whose direction of splicing was opposite in DDX5,17 KD cells to that in MBNL1 KD cells (orange) or the same (gray). The AS events were categorized into these two groups if PSI ≥ 5% and P < 0.05 in Student’s t test
Fig 4: DDX5,17 affect MBNL1 activity by modulating the ratio of MBNL1 splicing isoforms. A Genome browser view (hg38) of RNA-seq AS analyses showing the AS pattern of MBNL1 e5 and e7 in DDX5,17 KD HEK293T cells (n = 3; [46]), DDX5 KD K562 cells (n = 3; [38]) and MBNL1 KD HFE-145 cells (n = 3; [64]. B Representative gels and calculations of RT-PCR analyses of MBNL1 and MBNL2 AS events in siDDX5,17-treated HeLa cells. Data represent the mean ± SD (n = 3). Statistical significance was calculated in reference to siCtrl using Student’s t test; ns nonsignificant, ∗∗ for P < 0.01, ∗∗∗ for P < 0.001. C Graphical representation of distinct MBNL1 alternative splicing isoform pre-mRNAs differing in the presence of AS events and considered in this study. Numerical naming of each MBNL1 isoform: 40, 41, 42, 43, correspond to a molecular mass of the proteins in kDa and followes [23]. D Representative western blot analyses show different distributions of MBNL1 protein isoforms between control conditions and DDX5,17 KD HeLa cells. VINCULIN serves as a loading control. E A scheme of a fragment of MBNL1 pre-mRNA with a depicted position of binding of an antisense oligonucleotide (SSO-e7) complementary to a region across the 3’ss of e7 marked in red. F Representative gels and calculations of RT-PCR analyses of MBNL1 e5 and e7 upon SSO-e7 treatment in HSkM cells. Data represent the mean PSI values (n = 3). Statistical significance was calculated in reference to SSO-Ctrl using Student’s t test; ns nonsignificant, ∗∗∗ for P < 0.001. G Representative western blot analyses show changes in distribution of MBNL1 protein isoforms upon SSO-e7 treatment in HSkM. Numbers 40–43 correspond to a molecular mass of the MBNL1 proteins and refer to MBNL1 isoform pre-mRNAs illustrated in C. VINCULIN serves as a loading control. H Total level of MBNL1 upon SSO-e7 treatment in HSkM, as defined by western blotting and normalized to VINCULIN. Data represent the mean values ± SD (n = 3). Statistical significance was calculated in reference to SSO-Ctrl using Student’s t test; ns nonsignificant. I Representative gels and calculations of RT-PCR analyses of AS events shared by MBNL1 and DDX5,17 upon SSO-e7 treatment in HSkM. Six out of 17 analyzed events (PICALM e18, SPTAN1 e23, KTN1 e40, CLASP1 e25, CSDA e6, ACAD10 e6) were sensitive to SSO-e7 treatment and showed splicing changes that mimicked those observed in DDX5,17-depleted myoblasts (Fig. 2H, Suppl. Figure S2B). The left and right columns correspond to AS events sensitive and nonresponsive to SSO-e7 treatment, respectively. Data represent the mean PSI values (n = 3). Statistical significance was calculated in reference to SSO-Ctrl using Student’s t test; ns nonsignificant, ∗∗ for P < 0.01, ∗∗∗ for P < 0.001
Fig 5: DDX5,17 modulate AS of certain shared events independently of MBNL1,2. A Representative western blot analyses show protein levels in wild-type (WT) and Mbnl1,2 DKO MEFs upon Ddx5,17 depletion. Vinculin serves as a loading control. Blot images of all samples and calculated protein levels are shown in Suppl. Figure S6A. All samples derive from the same experiment and the blots were processed in parallel. B Representative gels of RT-PCR analyses of MBNL-regulated events whose splicing direction was either unchanged in DDX5,17 KD and Mbnl1,2 DKO cells (Ktn1 e38) or significantly deviated compared to that in control cells (Nfix e7, Exoc1 e12, Add3 e14). The image presents juxtaposed lanes that were non-adjacent on the gel. The lanes separation is delineated by a black separation. All samples derive from the same experiment and the bands were processed in parallel. C Calculation of RT-PCR analyses presented in B. Data represent the mean PSI values ± SD (n = 3). Statistical significance was calculated in reference to siCtrl using Student’s t test; ns nonsignificant, ∗ for P < 0.05, ∗∗∗ for P < 0.001. D A scheme of two Nfix e7 splicing minigenes, Nfix-BS and Nfix-mtBS, which carry MBNL-binding sites within an exon and upstream intron or their mutations, respectively [60]. MBNL BS, MBNL-binding site; MBNL mtBS, MBNL-binding sites carrying mutations; alt exon, alternative exon; const. exon, constitutive exon. E Representative gels and calculations of RT-PCR analyses of the Nfix e7 minigene splicing response in DDX5,17-depleted HeLa cells. Data represent the mean PSI values ± SD (n = 3). Statistical significance was calculated in reference to siCtrl using Student’s t test; ns nonsignificant, ∗∗∗ for P < 0.001
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