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

Journal: Biochemical and biophysical research communications

Article Title: PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

doi: 10.1016/j.bbrc.2022.08.090

Figure Lengend Snippet: Fig. 1. Plasmids of PRPF31 and PRPH2 were successfully transfected into HEK293T cells. A. PRPH2-mcherry. B Control-mcherry. C. PRPF31-eGFP. D. Control-eGFP. E. Immunoblotting showed both PRPF31-Flag and PRPH2-mcherry were detected. The length of the scale bars is 200 mm.

Article Snippet: ARPE-19 cells were cultured and transfected with and PRPH2mCherry vector for 24 h. Then cells were fixed and incubated with anti-PRPF31 antibody (Santa Cruz, TX) or IgG overnight.

Techniques: Transfection, Control, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

doi: 10.1016/j.bbrc.2022.08.090

Figure Lengend Snippet: Fig. 2. PRPF31 interacts with PRPH2 in HEK293T cells (A, B) and ARPE-19 cells (C) via Co-immunoprecipitation (Co-IP) assay. CoIP was performed using anti-Flag antibody (a) or anti-PRPH2 antibody (b, c). IB, immunoblotting, 3xFlag, Empty vector for PRPF31 expression vector, mCherry, empty vector for PRPH2-mCheryy expression vector.

Article Snippet: ARPE-19 cells were cultured and transfected with and PRPH2mCherry vector for 24 h. Then cells were fixed and incubated with anti-PRPF31 antibody (Santa Cruz, TX) or IgG overnight.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Expressing

Journal: Biochemical and biophysical research communications

Article Title: PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

doi: 10.1016/j.bbrc.2022.08.090

Figure Lengend Snippet: Fig. 3. PRPF31 and PRPH2 can partially co-localize in HEK293T cells transfected with PRP31-eGFP and PRPH2-mcherry (AeD), as well as in ARPE-19 cells transfected with PRPH2- mcherry and stained with Anti-PRPF31 antibody (IeL) or IgG control (EeH). The length of the scale bars is 10 mm.

Article Snippet: ARPE-19 cells were cultured and transfected with and PRPH2mCherry vector for 24 h. Then cells were fixed and incubated with anti-PRPF31 antibody (Santa Cruz, TX) or IgG overnight.

Techniques: Transfection, Staining, Control

Journal: Biochemical and biophysical research communications

Article Title: PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

doi: 10.1016/j.bbrc.2022.08.090

Figure Lengend Snippet: Fig. 4. Immunohistochemistry showed that PRPF31 and PRPH2 partially co-localized in the photoreceptor of C57BL/6J mice. The green channel represents PRPF31, the red channel represents PRPH2, and the blue channel represents DAPI. The upper panel is IgG control without the primary antibodies, and the lower panel is with primary antibodies. The length of the scale bars is 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: ARPE-19 cells were cultured and transfected with and PRPH2mCherry vector for 24 h. Then cells were fixed and incubated with anti-PRPF31 antibody (Santa Cruz, TX) or IgG overnight.

Techniques: Immunohistochemistry, Control

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, 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