Journal: Journal of Clinical Medicine

Article Title: Mesoporous Silica-Based Nanoparticles as Non-Viral Gene Delivery Platform for Treating Retinitis Pigmentosa

doi: 10.3390/jcm11082170

Figure Lengend Snippet: Transfection efficacy of PRPF31 gene using the nanoparticle delivery system PRPF31 -GFP/N-MSiNPs in a human cell line. Panels ( a – g ), immunofluorescence images of HEK-293 cells transfected with N-MSiNPs loaded with the PRPF31 -GFP plasmid. GFP fluorescent signal ( a , d ), in green; anti-PRPF31 fluorescent signal ( e ), in red. Cell nuclei were stained with DAPI ( b , f ), in blue. Merged images of the different channels are shown in ( c , g ). ( h ) WB of HEK-293 transfected cells showing the expression levels of the PRPF31 transgene fused to GFP. Protein extracts of HEK-293 treated with only Lipofectamine (negative control), in lane 1; from cells transfected with PRPF31 -GFP using Lipofectamine, in lane 2; from cells incubated with empty N-MSiNPs, in lane 3; and from cells transfected with N-MSiNPs loaded with PRPF31 -GFP, in lane 4. WBs were performed with anti-GFP (upper panel) and anti-PRPF31 (central panel) antibodies. GAPDH was used as a loading control (bottom panel). The (*) in the upper and central panels marks the expected molecular weight (80 kDa) of the PRPF31-GFP fused protein visualized by anti-GFP and ant-PRPF31 antibodies. The (#) in the central panel marks the expected molecular weight (55 kDa) of the endogenous PRPF31 protein. Scale bars represent 25 and 50 µm.

Article Snippet: Incubation with primary antibodies anti-PRPF31 (1:100; OriGene Technologies Inc., TA302582) and anti-GFP (1:200; Cell signaling 2956) was performed for 1 h at room temperature.

Techniques: Transfection, Immunofluorescence, Plasmid Preparation, Staining, Expressing, Negative Control, Incubation, Control, Molecular Weight

Journal: Journal of Clinical Medicine

Article Title: Mesoporous Silica-Based Nanoparticles as Non-Viral Gene Delivery Platform for Treating Retinitis Pigmentosa

doi: 10.3390/jcm11082170

Figure Lengend Snippet: Fundus images and the retinal section of PRPF31 -GFP/N-MSiNP subretinally injected mice. ( a ) Fundus image. ( b ) Fluorescence fundus. ( c ) Detail of the injection site showing GFP fluorescent dots. Immunostaining of retinal sections for GFP ( d ), in green; PRPF31 ( e ), in red. Nuclei stained with DAPI ( f ), in blue. ( g ) Merged images of the different channels. GFP and PRPF31 signal co-localized mainly in RPE cells. RPE = retinal pigment epithelium, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bar represents 50 µm.

Article Snippet: Incubation with primary antibodies anti-PRPF31 (1:100; OriGene Technologies Inc., TA302582) and anti-GFP (1:200; Cell signaling 2956) was performed for 1 h at room temperature.

Techniques: Injection, Fluorescence, Immunostaining, Staining

Journal: The Journal of Biological Chemistry

Article Title: Systematic proteomics of endogenous human cohesin reveals an interaction with diverse splicing factors and RNA-binding proteins required for mitotic progression

doi: 10.1074/jbc.RA119.007832

Figure Lengend Snippet: Cohesin-interacting splicing factors are required for mitotic progression. A, validation of siRNAs targeting representative cohesin-interacting splicing factors in HeLa cells. HeLa cells were transfected with negative control siRNA (s4390843) or gene-specific siRNAs as indicated by the Ambion catalogue number, cultured for 72 h, and harvested in RIPA buffer, and Western blotting was performed with the antibodies indicated. B, HeLa-H2B-GFP cells were transfected with siRNAs for SNRNP200 (ss22846), EFTUD2 (s17862), PRPF31 (s25123), SF3B3 (s23849), and sororin (s535461) and studied by live-cell imaging as described under “Experimental procedures.” Representative aberrant mitoses in siRNA-transfected cells are shown. Bar = 10 μm. For time-lapse movies with images every 5 min from 36 to 72 h post-transfection, see Movies S1 and S2. C, quantification of aberrant mitoses in siRNA-transfected cells. Cells in four fields were counted over the 36-h period of observation for each siRNA. The data shown are from three independent biological replicates. Error bars indicate standard deviation.

Article Snippet: Primary antibodies for immunoblotting were FLAG (M2) and tubulin (DM1α) from Sigma; sororin (ab192237) from Abcam; STAG2 (sc-81852), WAPL (sc-365189), HSPA8 (sc-7298), MAU2 (sc-243420), and NIPBL (sc-374625) from Santa Cruz Biotechnology; cyclin B1 (12231), cyclin E1 (20808), glyceraldehyde-3-phosphate dehydrogenase (2118), histone H3 (14269), and phospho-H3 (53348) from Cell Signaling Technologies; and ADAR1 (A303-884), DDX47 (A302-977), EFTUD2 (A300-957), HNRNPH (A300-511), PDCD11 (A303-804), PDS5A (A300-088), PDS5B (A300-538), PRPF31 (A303-919), RAD21 (A300-080), RBM10 (A301-006), RBM15 (A300-821), SF3B1 (A300-997), SF3B3 (A302-508), SMC1A (A300-055), SMC3 (A300-060), SNRNP200 (A303-453), and STAG1 (A302-579) from Bethyl Laboratories.

Techniques: Transfection, Negative Control, Cell Culture, Western Blot, Live Cell Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

doi: 10.1101/232397

Figure Lengend Snippet: (A) Gel electrophoresis showing the presence of a long mutant transcript (LM) isoform for the exon 11 deletion in patient-specific cells. The short mutant (SM) isoform is present only upon inhibition of NMD with puromycin (indicated by +); (B) The bar graph shows wild type PRPF31 mRNA in patient cells relative to controls from (A). Data are representative of at least 3 independent repeats, RO – retinal organoids; (C-D) Wild type PRPF31 is significantly reduced in patient RPE cells and less notably in 3D retinal organoids. The LM form and reduced SART1 is observed only in the patient RPE cells; (E) The bar graph shows wild type PRPF31 levels in patient cells relative to normal cells quantified from (C); (F) Patient RPE cells and 3D retinal organoids exhibit a notable defect in the alternative splicing of E1A mini-gene reporter. Schematic representation of alternative splice variants of the E1A reporter (F) and Denaturing PAGE and autoradiography using a phosphoimager (G); (H) Northern Blot analysis showing the level of snRNAs in various normal and patient cells. Total RNA was isolated from each sample and snRNA levels were analysed by denaturing PAGE followed by Northern blotting using probes against U1, U2, U4, U5, U6 and 5S rRNA (top). The levels of snRNAs were quantified and normalized to the amount of 5S rRNA (bottom).

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: RECOVERIN (Millipore, 1:800), NRL (Santa Cruz, 1:800), CRALBP (Abcam, 1:500), HuC/D (Invitrogen, 1:500), PKCα (BD Pharmingen, 1:500), ARL13B (Proteintech, 1:500), PRPF31 (Abnova, 1:500), SNRPB Monoclonal Antibody (Y12) (Thermo, 1:500).

Techniques: Nucleic Acid Electrophoresis, Mutagenesis, Inhibition, Alternative Splicing, Autoradiography, Northern Blot, Isolation

Journal: bioRxiv

Article Title: Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

doi: 10.1101/232397

Figure Lengend Snippet: (A) rMATS analysis showing that RP11 RPE have the highest percentage of transcripts containing retained introns (RI) and alternative 3’ splice sites (A3SS); (B, C) Gene Ontology enrichment analysis showing biological and cellular processes affected by alternative splicing respectively; (D, E) Gene Ontology enrichment analysis showing biological and cellular processes affected by alternative splicing respectively in the Prpf31 +/− retinae; (F, G) Validation of RNA-seq findings by semi-quantitative RT-PCR, showing alternative splicing events in the key genes involved in pre-mRNA processing, cell adhesion and cilia. Blue numbers under lanes indicate the percentage spliced in (PSI) values for the major isoform. Loading control is for GAPDH.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: RECOVERIN (Millipore, 1:800), NRL (Santa Cruz, 1:800), CRALBP (Abcam, 1:500), HuC/D (Invitrogen, 1:500), PKCα (BD Pharmingen, 1:500), ARL13B (Proteintech, 1:500), PRPF31 (Abnova, 1:500), SNRPB Monoclonal Antibody (Y12) (Thermo, 1:500).

Techniques: Alternative Splicing, RNA Sequencing Assay, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

doi: 10.1101/232397

Figure Lengend Snippet: (A) PRPF31 siRNA knockdown in human hTERT-RPE1 cells causes a significant decrease in cilia incidence (lower left) and PRPF31 protein levels (lower right) compared to scrambled negative control (siScr) siRNA; (B) Gli1 reporter assays of Shh activity measured in NIH3T3-GL cells following knockdown for Ptch1 (positive control), scrambled negative control siRNA (siScr) and Prpf31. Cells were treated with either 100nM SAG or vehicle control for 48hr, as indicated. Assays results are expressed in arbitrary units of the ratio of firefly: Renilla luciferase activities; (C) Ciliary localization of IFT88 (green) in primary cilia of hTERT-RPE1 cells (visualized by staining for γ-tubulin and poly-glutamylated tubulin; red) showing mis-localization of IFT88 (arrowheads) at ciliary tips following PRPF31 knockdown. Bar graph quantitates the percentage of cilia with IFT88 at their tip. Scale bar = lμm; (D) Visualization and quantitative analysis of the transition zone protein CC2D2A (green) and ARL13B (red); (E) Visualization and quantitative analysis of the transition zone (TZ) protein RPGRIP1L (green) and cilia (y-tubulin and poly-glutamylated tubulin; red) showing mis-localization of RPGRIP1L from the TZ into the ciliary axoneme (arrowheads) following PRPF31 knockdown. (F-G) Ciliary localization of IFT88 and RPGRIP1L (green) in RP11 RPE cells showing mis-localization of IFT88 (arrowheads) at ciliary tips and RPGRIP1L from the TZ into the ciliary axoneme (arrowheads). A-G: Data shown as mean ± SEM. Pair-wise statistical comparisons are indicated by braces: n.s. no significance.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: RECOVERIN (Millipore, 1:800), NRL (Santa Cruz, 1:800), CRALBP (Abcam, 1:500), HuC/D (Invitrogen, 1:500), PKCα (BD Pharmingen, 1:500), ARL13B (Proteintech, 1:500), PRPF31 (Abnova, 1:500), SNRPB Monoclonal Antibody (Y12) (Thermo, 1:500).

Techniques: Knockdown, Negative Control, Activity Assay, Positive Control, Control, Luciferase, Staining

Journal: bioRxiv

Article Title: Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

doi: 10.1101/232397

Figure Lengend Snippet: (A, B, C) CRISPR/Cas9 correction of the PRPF31 mutation in exon 11; (D, E) Quantification of cilia length and incidence in PRPF31-edited RPE. (F) TEM analysis of PRPF31-e dited RPE cilia showing morphologically normal cilia; (G) Increased phagocytosis in PRPF31-e dited RPE. (H, I, J) Restoration of apical-basal polarity in PRPF31-e dited RPE. (K, L) Quantification of cilia length and frequency in PRPF31-edited photoreceptors; (M) TEM analysis of PRPF31-edited photoreceptor cilia showing morphologically normal cilia. C-E, G-I, K, L: Data shown as mean ± SEM, n=3. Statistical significance of pair-wise comparisons is indicated by n.s. not significant; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 (Student’s paired t-test).

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: RECOVERIN (Millipore, 1:800), NRL (Santa Cruz, 1:800), CRALBP (Abcam, 1:500), HuC/D (Invitrogen, 1:500), PKCα (BD Pharmingen, 1:500), ARL13B (Proteintech, 1:500), PRPF31 (Abnova, 1:500), SNRPB Monoclonal Antibody (Y12) (Thermo, 1:500).

Techniques: CRISPR, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: Expression patterns of prpf31 during zebrafish embryonic development in siblings and prpf31 −/− zebrafish. A – L and A’ – L’ , prpf31 expression in siblings and prpf31 −/− zebrafish was examined by WISH at different developmental stages. E – L and E’ – L’ : lateral views, anterior to the left and dorsal upward. G and G’ : magnified views of the ICM in ( E and E’ ). H and H’ : magnified views of the PBI in ( F and F’ ). K , L , K’ , and L’ : magnified views of the CHT in ( I , J , I’ , and J ’). The number of embryos with similar gene expression patterns among all embryos examined was shown at the bottom right of each panel. The scale bars represent 250 μm. CHT, caudal hematopoietic tissue; HSPC, hematopoietic stem and progenitor cell; ICM, intermediate cell mass; PBI, posterior blood island; PRPF, pre-mRNA processing factor; WISH, whole-mount in situ hybridization.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Expressing, Gene Expression, In Situ Hybridization

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: Definitive hematopoiesis is impaired in prpf31 −/− zebrafish. A , bright field observation showed morphological abnormalities in prpf31 −/− zebrafish at 3 dpf. WISH manifested the expression of HSPC markers runx1 and cmyb were noticeably reduced and rescue of cmyb expression after prpf31 mRNA injection. Lateral views. B , expression of the myelocyte markers lyz , l-plastin , and mpx by WISH, and staining signal of Sudan black B labeled neutrophils were significantly decreased in the CHT of prpf31 −/− zebrafish at 3 dpf. Lateral views. C , expression of the erythrocyte markers hbae1.1 , hbae3 , and hbbe1 were almost undetectable in the CHT of prpf31 −/− zebrafish at 4 dpf, although there was only slightly decrease compared with siblings at 3 dpf by WISH. Lateral views. D , expression of the early T cell marker rag1 was almost completely absent in the thymus of prpf31 −/− zebrafish at 3 dpf by WISH. Lateral and dorsal views. Black arrows denote the CHT region. Red arrows denote the thymus region. The number of embryos with similar gene expression patterns among all embryos examined was shown at the bottom right of each panel. The scale bars represent 250 μm. E , quantification of mRNA signals of prpf31 −/− zebrafish and siblings detected by WISH in ( A – D ). The total number of embryos examined was indicated below each column. Mean ± SD; unpaired two-tailed t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. CHT, caudal hematopoietic tissue; dpf, days postfertilization; hpf, hours postfertilization; HSPC, hematopoietic stem and progenitor cell; PRPF, pre-mRNA processing factor; WISH, whole-mount in situ hybridization.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Expressing, Injection, Staining, Labeling, Marker, Gene Expression, Two Tailed Test, In Situ Hybridization

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: HSPC expansion and maintenance in the CHT are compromised in prpf31 −/− zebrafish. A – I and A ’– I’ , time-course analysis of the expression of HSPC markers runx1 and cmyb in prpf31 −/− and sibling embryos from 28 hpf to 48 hpf by WISH. The reduced expression of cmyb in prpf31 −/− zebrafish was rescued by prpf31 mRNA injection at 48 hpf. Lateral views. Red arrows denote the AGM region. Black arrows denote the CHT region. The number of embryos with similar gene expression patterns among all embryos examined was shown at the bottom right of each panel. The scale bars represent 250 μm. J , quantification of mRNA signals of prpf31 −/− zebrafish and siblings detected by WISH in ( A – I and A’ – I’ ). The total number of embryos examined was indicated below each column. Mean ± SD; unpaired two-tailed t test; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, not significant. K , in vivo observation of HSPCs using transgenic line Tg ( cmyb : EGFP) of prpf31 −/− zebrafish and siblings, in the AGM and CHT at 36 hpf, 48 hpf, and 3 dpf. The total number of embryos examined was indicated at the bottom right of each panel. The scale bars represent 50 μm. L , quantification of the number of HSPCs in the AGM and CHT of prpf31 −/− zebrafish and siblings observed in ( K ). The total number of embryos examined was indicated below each column. Mean ± SD; unpaired two-tailed t test; ∗ p < 0.05, ∗∗∗ p < 0.001; ns, not significant. AGM, aorta-gonad-mesonephros; CHT, caudal hematopoietic tissue; dpf, days postfertilization; hpf, hours postfertilization; HSPC, hematopoietic stem and progenitor cell; PRPF, pre-mRNA processing factor; WISH, whole-mount in situ hybridization. EGFP, enhanced green fluorescent protein.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Expressing, Injection, Gene Expression, Two Tailed Test, In Vivo, Transgenic Assay, In Situ Hybridization

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: HSPC proliferation in the CHT of prpf31 −/− zebrafish is arrested in M phase. A , double staining of cmyb :EGFP and EdU showed no obvious difference of EdU + HSPCs in the AGM and CHT at 36 and 48 hpf. The total number of embryos examined were indicated at the top right of each panel. The scale bars represent 50 μm. B , quantification of the percentage and number of EdU + HSPCs was detected in ( A ). The total number of embryos examined was indicated below each column. Mean ± SD; unpaired two-tailed t test; ∗ p < 0.05, ∗∗ p < 0.01; ns, not significant. C , double immunostaining of cmyb :EGFP and pH3 (Ser10) showed a significant increase of pH3 + HSPCs in the CHT at 48 hpf, but not in the AGM and CHT at 36 hpf. The total number of embryos examined were indicated at the top right of each panel. The scale bars represent 50 μm. D , quantification of the percentage and number of pH3 + HSPCs was detected in ( C ). The total number of embryos examined was indicated below each column. Mean ± SD; unpaired two-tailed t test; ∗ p < 0.05, ∗∗∗ p < 0.001; ns, not significant. AGM, aorta-gonad-mesonephros; CHT, caudal hematopoietic tissue; EdU, 5-ethynyl-29-deoxyuridine; hpf, hours postfertilization; HSPC, hematopoietic stem and progenitor cell; pH3, phospho-histone 3. EGFP, enhanced green fluorescent protein.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Double Staining, Two Tailed Test, Double Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: prpf31 −/− zebrafish exhibit severe aberrant alternative splicing of mitosis-related genes. A , DASEs corresponding to three representative GOBP terms, that is chromosome organization (GO: 0051276), regulation of cell cycle (GO: 0051726), and microtubule-based process (GO: 0007017) were visualized by heatmaps of normalized PSI. B , the DASEs of the representative genes were confirmed by SqRT-PCR. Data were shown as mean ± SD of three independent experiments (n = 3); unpaired two-tailed t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. DASE, differential alternative splicing event; PRPF, pre-mRNA processing factor; PSI, percent spliced in; SqRT-PCR, semiquantitative reverse transcription PCR. GOBP, Gene Ontology biological processes.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Alternative Splicing, Two Tailed Test, Reverse Transcription

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: Aberrant alternative splicing of mitosis-related genes leads to mitotic malformations of HSPCs in the CHT of prpf31 −/− zebrafish. A , quadruple staining of DAPI, cmyb :EGFP, pH3 (Ser10), and tubulin showed the mitotic status of HSPCs in the CHT of prpf31 −/− zebrafish and siblings at 36 and 48 hpf. At least 25 mitotic HSPCs in more than six embryos were observed for each group. The scale bars represent 4 μm. B , expression of four aberrantly spliced mitosis-related genes ( septin6 , smarcb1b , tinf2 , and usp22 ) in the CHT of siblings and prpf31 −/− zebrafish at 36 and 48 hpf by WISH. Lateral views. Black arrows denote the CHT region. The number of embryos with similar gene expression patterns among all embryos examined were shown on the top right of each panel. The scale bars represent 100 μm. C , qRT-PCR analysis of four aberrantly spliced mitosis-related genes ( septin6 , smarcb1b , tinf2, and usp22 ) in siblings and prpf31 −/− zebrafish at 36 and 48 hpf. Data were shown as mean ± SD of three independent experiments (n = 3); unpaired two-tailed t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001; ns, not significant. CHT, caudal hematopoietic tissue; hpf, hours postfertilization; HSPC, hematopoietic stem and progenitor cell; pH3, phospho-histone 3; qRT-PCR, quantitative real-time PCR; WISH, whole-mount in situ hybridization. DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Alternative Splicing, Staining, Expressing, Gene Expression, Quantitative RT-PCR, Two Tailed Test, Real-time Polymerase Chain Reaction, In Situ Hybridization

Journal: The Journal of Biological Chemistry

Article Title: The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

doi: 10.1016/j.jbc.2024.105772

Figure Lengend Snippet: Model of Prpf31 action in HSPC expansion during zebrafish embryogenesis. In zebrafish embryogenesis, the nascent HSPCs undergo extensive proliferation for pool expansion and commitment-proliferation-differentiation in the CHT, which demand rapid and periodic regulated pre-mRNA alterative splicing. Accurate and sequential regulated pre-mRNA alterative splicing ensure efficient and precise HSPCs mitosis for rapid blood replenishment. In prpf31 −/− zebrafish, the spliceosomes cannot assemble effectively due to deficiency of Prpf31, which compromises the splicing efficiency of the spliceosome machine, and results in aberrant mRNA alternative splicing, perturbs the alternative splicing of mitosis-related genes, predisposes HSPCs to malformed mitosis and cell cycle arrest in M phase, and eventually impaired the expansion and differentiation of HSPCs in the CHT. CHT, caudal hematopoietic tissue; HSPC, hematopoietic stem and progenitor cell; PRPF, pre-mRNA processing factor.

Article Snippet: The following primary antibodies were used: anti-PRPF31 antibody (GeneTex, GTX117081, 1:1000), anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3000), and anti-phospho-histone H3 (Ser10) antibody (Affinity, AF3358, 1:1000).

Techniques: Alternative Splicing