Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: U2AF2/circ_0036763/miR-583/ACAN axis is associated with IDD development . (A) The expression of circ_0036763 in HNPCs isolated from IDD patients and normal HNPCs was evaluated by qRT-PCR. (B) The putative interaction between circ_0036763 and miR583 was predicted by bioinformatic analysis. (C) The expression of miR583 in HNPCs isolated from IDD patients and normal HNPCs was evaluated by qRT-PCR. (D) The expression of U2AF2 in HNPCs isolated from IDD patients and normal HNPCs was evaluated by WB. (E) The putative interaction between miR-583 and ACAN was predicted by bioinformatic analysis. (F) The expression of ACAN in HNPCs isolated from IDD patients and normal HNPCs was evaluated by Western blot. (G) HNPCs isolated from IDD patients were transfected with circ_0036763 OE or the empty vector (vec), and the expression of ACAN, Collagen I and Collagen II was evaluated by WB. ∗ P < 0.05, ∗∗ P < 0.01 and ns indicates no significant difference.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: The RBP-circRNA interaction between circ_0036763 and U2AF2.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques:

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: U2AF2 promotes the mature of circ_0036763 . (A) The direct interaction between pre-circ_0036763 and U2AF2 was determined by RIP assay. (B) The three siRNAs targeting U2AF2 were transfected into HNPCs, and the transfection efficiency was confirmed by qRT-PCR. (C) The overexpressing plasmid for U2AF2 (U2AF2 OE) and si-U2AF2 were transfected into HNPCs, and the expression of circ_0036763 was evaluated by qRT-PCR. (D) The overexpressing plasmid for U2AF2 (U2AF2 OE) was transfected into HNPCs isolated from IDD patients, and the expression of circ_0036763 was evaluated by qRT-PCR. (E) The level of pre-circ_0036763 in HNPCs isolated from IDD patients and normal HNPCs detected by qRT-PCR. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 and ns indicates no significant difference.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Transfection, Quantitative RT-PCR, Plasmid Preparation, Expressing, Isolation

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: U2AF2 from bMSCs-derived exosomes regulates expressions of circ_0036763 and ACAN in HNPCs isolated from IDD patients . (A and B) The level of U2AF2 in HNPCs and bMSCs-derived exosomes was evaluated by qRT-PCR (A) and WB (B). (C) BMSCs or bMSCs-derived exosomes were co-cultured with HNPCs isolated from IDD patients. When needed, exosomes inhibitor G4869 was added. The expression of U2AF2 in HNPCs isolated from IDD patients was evaluated by WB. (D and E) BMSCs-derived exosomes, or si-U2AF2 transfected bMSCs were co-cultured with HNPCs isolated from IDD patients. (D) The level of circ_0036763 in HNPCs isolated from IDD patients was evaluated by qRT-PCR. (E) The expression of ACAN in HNPCs isolated from IDD patients was evaluated by Western blot. ∗∗ P < 0.01.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Derivative Assay, Isolation, Quantitative RT-PCR, Cell Culture, Expressing, Transfection, Western Blot

Journal: Heliyon

Article Title: LncRNA CECR7 boosts hepatocellular carcinoma progression by recruiting RNA binding protein U2AF2 to enhance the stability of EXO1 mRNA

doi: 10.1016/j.heliyon.2023.e19862

Figure Lengend Snippet: CECR7 stabilize EXO1 mRNA under the mediation of U2AF2 protein. (A) The platform starBase ( http://starbase.sysu.edu.cn ) was applied to predict the potential RNA binding protein for both CECR7 and EXO1 mRNA. ( B, C ) RIP assay using antibody against U2AF2 to determine the enrichment of CECR7 and EXO1 mRNA in HCC cells (Mean ± SD; n = 3). ** P < 0.01, *** P < 0.001, Student's t -test. ( D, E ) RT-qPCR analysis and Western blot were used to detect the effect of CECR7 on U2AF2 expression. ( F, G ) U2AF2-overexpressing reversed the repression of EXO1 mRNA by CECR7 shRNA#1, and the EXO1 mRNA induction induced by CECR7-overexpressing was abrogated by U2AF2 shRNA (Mean ± SD; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA.

Article Snippet: Lysates of HCC cells were cultured in RIP containing magnetic beads conjugated with antibodies against U2AF2 (NBP2-33397, Novus, USA) or control IgG (NBP2-34250, Novus, USA) for 6h at 4 °C.

Techniques: RNA Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, shRNA

Journal: Nature medicine

Article Title: H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers

doi: 10.1038/nm.4493

Figure Lengend Snippet: H3B-8800 inhibits splicing of short, GC-rich introns and affects splicing of mRNAs encoding spliceosome components. (a) Hexbin plot showing relative abundance of introns based on length (y axis) and GC content (x axis) of all introns of expressed genes in K562 cells (background, left) versus introns retained in SF3B1K700E cells after H3B-8800 treatment (13 nM, right). The percentage of the total intron count contained within each hex is indicated by its color. (b) GC content within introns retained after H3B-8800 treatment of K562 SF3B1K700E cells (red; n = 5,788 introns) versus a randomly selected subset of introns from background (black; n = 10,000 introns). The GC content within the exons that followed is also shown. The solid line represents the mean GC content, and the shaded area surrounding the solid line represents the 95% confidence interval of that measurement. (c) The 3′ splice site motif of introns retained in K562 SF3B1K700E cells following H3B-8800 treatment as compared to background. (d) Branchpoint sequence motif (extending from 6 nt upstream of the branchpoint nucleotide to 4 nt downstream of the branchpoint) representing all branchpoints sequenced by Mercer et al.26 (left) and only those branchpoints within introns retained in in K562 SF3B1K700E cells following H3B-8800 treatment (right). (e) Top, depiction of two Ad2-derived pre-MRNA substrates: Ad2.14, in which the Py-tract contains five cytosines and five thymines, and Ad2.17, which also contains an alteration in the BPS to reduce binding affinity to U2 snRNP. The branchpoint nucleotide is labelled in red, and the nucleotide that was changed between Ad2.14 and Ad2.17 to alter branchpoint strength is bolded. Bottom, the results from in vitro splicing using these two pre-mRNA substrates. The y axis represents the percent response relative to DMSO control (0%). Data are represented as mean ± s.d.; n = 3 technical replicates. (f) Sashimi plot representing the average read density of three technical replicates in introns 3 and 4 of U2AF2 (top) and introns 20, 21, and 22 of RBM10 (below) of RNA-seq samples from K562 SF3B1K700E cells treated with DMSO or H3B-8800. Exons are shown in black, and introns are shown in blue (DMSO treated) or red (H3B-8800 treated). Relative GC content (derived using a 100-bp sliding window) is depicted in a heat map below the reference sequence. (g) The effect of an increasing concentration of H3B-8800 on the level of U2AF2 pre-mRNA (black squares) and mature mRNA (open squares) in K562 SF3B1K700E cells as quantified by qPCR. Data are represented as mean ± s.e.m.; n = 3 technical replicates. (h) Western blot analysis of U2AF2, SRPK1, and MCL1 protein levels following treatment of K562 SF3B1K700E cells with H3B-8800 or E7107 at the indicated concentrations. β-actin and GAPDH were used as loading controls.

Article Snippet: Membranes were blocked in Odyssey Blocking Buffer (LiCor) or 5% milk in Tris-buffered saline and 0.1% Tween20 and were probed overnight at 4 °C with antibodies against the following targets: U2AF2 (1:5,000, Sigma, U4758), HA-Tag (1:1,000, Abcam, 18181), SRPK1 (1:1,000, Bethyl, A302–461A), MCL1 (1:1,000, Cell Signaling Technologies, 5453), β-Actin (1:1,000; Sigma-Aldrich, A5441) or GAPDH (1:5,000, Sigma, G8795 and G9545).

Techniques: Sequencing, Derivative Assay, Binding Assay, In Vitro, RNA Sequencing Assay, Concentration Assay, Western Blot

Journal: Genome Research

Article Title: RNA structure replaces the need for U2AF2 in splicing

doi: 10.1101/gr.181008.114

Figure Lengend Snippet: The splicing of (AC) m -(GT) n introns requires components of U2 snRNP but not U2AF2. In vitro splicing substrates were prepared from the constructs used in and the model in vitro splicing construct Ad81. The in vitro–transcribed RNA was incubated in HeLa nuclear extract for the splicing assay. ( A ) The splicing of the Ad81 control was compared to the (AC) m -(GT) n intron with pre-incubation with either: no antibody, a control antibody, or increasing amounts of anti-U2AF2 antibody. The input pre-mRNA and the splicing products, including the lariat intermediate and free first exon from the first step of splicing, the free lariat, and ligated exon from the second step of splicing, were resolved on an 8M urea gel and visualized by autoradiography with a phosphoimager. ( B ) The comparison described above was repeated with antibody targeting SF3B1, a component of the U2 snRNP. ( C ) u2af2 knockdown (KD) in zebrafish embryos. The KD embryos developed without gross phenotypic defect by 48 h but with lower hatch rate. ( D ) Western blotting of U2af2 24 or 48 h after injection. Beta actin served as a loading control. ( E ) RT-PCR of single intron transcripts containing (AC) m -(GT) n repeats ( left ) or without the repeats ( right ). Pre-mRNA and/or spliced mRNA is depicted on the right of the gel images. A bracket indicates the smear of the PCR product, possibly due to the loss of splicing accuracy.

Article Snippet: Splicing reactions were prepared in the absence of the RNA substrate with 200 or 600 ng of blocking MC3 monoclonal antibody against U2AF2 (also known as U2AF65; Santa Cruz Biotechnology), 600 ng antibodies against SF3B3 (Proteintech), or 600 ng normal rabbit IgG (Santa Cruz Biotechnologies) as a control, incubated at 30°C for 10 min before supplementing the substrate, and allowing splicing at 30°C for 60 min.

Techniques: In Vitro, Construct, Incubation, Splicing Assay, Control, Autoradiography, Comparison, Knockdown, Western Blot, Injection, Reverse Transcription Polymerase Chain Reaction

Journal: Genome Research

Article Title: RNA structure replaces the need for U2AF2 in splicing

doi: 10.1101/gr.181008.114

Figure Lengend Snippet: Model for secondary structure-dependent splicing. ( Left ) Zebrafish (AC) m -(GT) n introns: 5′ ss and 3′ ss are brought closer by base-pairing of AC and GT repeats. This intronic bridging can override the requirement of U2AF2 for 3′ ss recognition. ( Right ) Human (GGG) m -(CCC) n introns: 16% of human introns contain multiple copies of complementary G and C triplets near 5′ ss and 3′ ss, respectively, that stabilize the intronic structure. They may bridge the splice sites and facilitate splicing as the zebrafish (AC) m -(GT) n introns.

Article Snippet: Splicing reactions were prepared in the absence of the RNA substrate with 200 or 600 ng of blocking MC3 monoclonal antibody against U2AF2 (also known as U2AF65; Santa Cruz Biotechnology), 600 ng antibodies against SF3B3 (Proteintech), or 600 ng normal rabbit IgG (Santa Cruz Biotechnologies) as a control, incubated at 30°C for 10 min before supplementing the substrate, and allowing splicing at 30°C for 60 min.

Techniques: