Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Sez6, Sez6L, and Sez6L2 are expressed by principal (excitatory, pyramidal) neurons of the mouse hippocampus at much higher levels than other known complement regulators (namely Crry, C4BP, CFH, C1-INH, DAF, and MCP). Expression data was obtained from Hipposeq: a comprehensive RNA-Seq database of gene expression in hippocampal principal neurons ( http://hipposeq.janelia.org ; ). The RNA samples used in this database were isolated from mouse hippocampal principal neurons micro-dissected from the CA1, CA3, or Dentate Gyrus (DG) cell layers of the hippocampus at Postnatal Day 25-32. Differential gene expression is shown in the heatmap with the relative units of FPKM (Fragments Per Kilobase of Exon Per Million Reads Mapped.) B) Brain sections from adult WT mice or Sez6 triple knockout mice (TKO) were immuno-stained for Sez6L2 (green) and DAPI and imaged in the CA1 region of the hippocampus. Scale Bar= 27μm. High density Sez6L2 staining occurs around cell bodies in the pyramidal layer, but significant Sez6L2 is also found in the stratum radiatum and stratum oriens. C) Higher magnification images of sections immuno-stained for Sez6L2 (green) and the postsynaptic protein, Homer1 (red), shows Sez6L2 is found near or co-localized with synapses in the stratum radiatum. Scale bar = 1.8μm.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Triple Knockout, Staining

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Purified Sez6L2-MH is shown by a coomassie stained gel and by western blot with anti-Sez6L2 and anti-Myc antibodies. Lanes with the coomassie stain are from the same gel. B) Schematic of Sez6L2 and Sez6L2-MH domain structures. CCP=Domain abundant in c omplement c ontrol p roteins. CCP domains are also known as SUSHI repeats or short complement-like repeat (SCR) CUB= Domains named after complement C 1r/C1s, u EGF, and B MP1 TM=Transmembrane region. Sez6L2-MH was made by replacing the transmembrane and cytoplasmic tail domains with a tandem Myc, 6xHis tag. C) and D) Classical pathway hemolysis assay. Antibody-coated sheep erythrocytes were exposed to human serum pre-incubated with purified Sez6L2-MH, C1-INH, FH, C4BP, H-DAF, or BSA. After 30 mins, the percent of cell lysis was measured by spectrophotometry (A415). C1-INH, FH, C4BP, and H-DAF are known complement inhibitors and were used as positive controls. BSA was used as a negative control for comparison. H-DAF is His-tagged DAF. 1-way ANOVA (P <0.0001; F(6,14)=314.4). N=3 (1 experiment with 3 replicates; Representative of 4 independent experiments with Sez6L2 and buffer and 2-3 experiments with each control protein). D) Supernatants from classical pathway hemolysis assays were further analyzed for C3 cleavage products by western blot. Supernatants in D are from same experiment shown in C. Sez6L2-MH increases the amount of the C3dg cleavage product similar to other complement regulators that work at the level of the C3 convertase. Sez6L2-MH runs just above the C3α band of C3. When large amounts of Sez6L2 are present it can cause C3α to run lower on the gel and sometimes mildly interferes with antibody binding to C3α. The membrane blotted for C3 was also stained with ponceau S to reveal total protein and shows the presence of the purified proteins in each sample. E) Schematic of C3 cleavage products. The C3d region recognized by our C3 antibody is highlighted in dark grey. F) Alternative pathway hemolysis assay. Rabbit erythrocytes were exposed to human serum pre-incubated with Sez6L2-MH, complement regulators, or BSA in presence of 12.5mM MgEGTA to block the classical pathway. Then the percent of cell lysis was measured by spectrophotometry (A415). 1-way ANOVA (P =0.0122; F(4,7)=7.298). Results are the mean of three independent experiments which tested 4 independent Sez6L2-MH preps multiple times. N=3 for buffer and Sez6L2-MH; N=2 for FH, H-DAF, and BSA. For all graphs * = p <0.05; ** = p<0.01

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Purification, Staining, Western Blot, Hemolysis Assay, Incubation, Lysis, Spectrophotometry, Negative Control, Binding Assay, Blocking Assay

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Schematic of Factor I and cofactor cleavage of C3b and iC3b. B) Factor I cleavage assay of C3b. C3b and Factor I (FI) were incubated alone, with Factor H (FH), or C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using antibodies that recognize C3d, a region within the C3α chain (and highlighted by the black rectangle in the schematics in (A)). Coomassie stained gels are also shown. FH and C4BP are known co-factors of FI towards C3b and served as positive controls. Incubation of C3b and FI with Sez6L2-MH also generated the C3 cleavage products α’1 and α’2 showing Sez6L2-MH is a cofactor for Factor I cleavage of C3b. C) Schematic of Factor I + cofactor cleavage of C4b. D) C4b and Factor I (FI) were incubated alone, with FH, C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using a C4 polyclonal antibody or coomassie stained gels. C4 components recognized by the C4 antibody are colored black in the schematic in C. C4BP is a known cofactor of FI for C4b cleavage and served as a positive control. Incubation of C4b and FI with Sez6L2-MH did not result in the appearance of C4b cleavage products.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Cleavage Assay, Incubation, Western Blot, Staining, Generated, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: Factor I cleavage assay of C3b (A) or C4b (B). C3b or C4b and Factor I (FI) were incubated alone, with Factor H (FH, 1ug), C4BP (1ug), or Sez6L2-MH (1ug or 5ug) for 4 or 8 hours at 37°C. Then samples were analyzed by coomassie stained gels. Sez6L2-MH aids significant factor I cleavage of C3b but not C4b at 4 and 8 hours resulting in appearance of the C3b cleavage products, C3α’1 and C3α’2, but not the C4b cleavage products, C4d and C4α3.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Cleavage Assay, Incubation, Staining

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A and B) Alternative C3 convertase assay: A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot (B). C) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2a, then incubated with Sez6L2-MH or H-DAF at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2a) was detected using an anti-C2 antibody ELISA. For A and C ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: 1-way ANOVAS with Holms-Sidak multiple comparison’s tests were performed for each complement inhibitor with comparisons to 0 ug/mL controls. * p<0.05.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Convertase Assay, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A-B) Sez6L2 inhibits C3 deposition at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5 depleted human serum for 1 hour and then labeled with anti-C3b/C3c antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. B) C3 deposition on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; 1 experiment with 3 replicates (representative of 3+ independent experiments). C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. D-I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. D) 5% Contour plots of C3 versus Myc fluorescence (top layer) and C3 fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as myc positive (top box) and myc negative (lower box) populations. For C3 histograms, dark grey, solid line population = Myc positive cells; Light grey, dotted line population= Myc negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. E) Quantification of the average median C3 fluorescence intensity from myc positive and myc negative cells within each sample. Statistics = t-tests. N=3 (1 experiment with 3 replicates; Representative of 4+ independent experiments). F) Average median C3 fluorescence intensities after normalization to the myc negative cells from each experimental group. ANOVA between Myc+ cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3 deposition at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. F) Average median Myc fluorescence intensity from myc positive cells ANOVA (p<0.001; F(4, 15)=36.79). G) Average % of Myc positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections F-H, N=4 (4 independent experiments). I) Sez6 blocks complement deposition more efficiently than Sez6L2 and Sez6L even when comparing similar levels of myc surface expression. Average C3 median fluorescence intensity normalized to internal myc negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the myc median fluorescence intensity. N=3 (1 experiment with 3 replicates, Representative of 3 independent experiments). For all graphs * = p <0.05; ** = p<0.01; # = p<0.001 for all Myc+ groups compared to M-CR2.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Transfection, Labeling, Flow Cytometry, Expressing, Fluorescence, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: Sez6 family FASTAs sequences were uploaded into the web-based program provided by Ojha et al ( http://coredo.nccs.res.in/meme-5.0.3/CoReDo/home.html ). Classical model complement regulatory proteins (CRPs) have motifs in the order of M5, M3, M1, M2, and either M4 or M1 spaced across 3 consecutive CCP domains. Sez6L2 has this same pattern, but Sez6 and Sez6L do not.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Representative images of PCIF1 staining in tumor and non-tumorous tissues from HNSCC patients (FAH-SYSU-Cohort1). Scale bars: 100 μm. ( B ) Quantification of PCIF1 staining score between tumor tissue samples ( n = 81) and non-tumorous tissue samples ( n = 57) from HNSCC patients (FAH-SYSU-Cohort1). *** P < 0.001 by 2-tailed unpaired Student’s t test. ( C ) Kaplan-Meier curve depicting the overall survival of patients with HNSCC (FAH-SYSU-Cohort1) stratified by PCIF1 expression levels. P values were calculated by log-rank test. ( D ) Representative images of PCIF1 staining in tumor and non-tumorous tissues from HNSCC patients (HS-SYSU-Cohort2). Scale bars: 100 μm. ( E ) Quantification of PCIF1 staining score between tumor tissue samples ( n = 40) and non-tumorous tissue samples ( n = 40) from HNSCC patients (HS-SYSU-Cohort2). *** P < 0.001 by 2-tailed unpaired Student’s t test. ( F ) Kaplan-Meier curve depicting the overall survival of patients with HNSCC (HS-SYSU-Cohort2) stratified by PCIF1 expression levels. P values were calculated by log-rank test.

Article Snippet: For PCIF1 overexpression plasmids, the full-length open reading frames of the human PCIF1 gene (NCBI Reference Sequence NM_022104.3) were cloned into the pLVX vector (GeneCopoeia).

Techniques: Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Western blotting analyses of the PCIF1 expression in the cell lines. ( B ) Western blotting analyses detecting the PCIF1 expression in SCC9 (left) and SCC25 (right) control cells and PCIF1-KO cells. ( C ) Cell Counting Kit-8 (CCK8) assay of cell viability in control and PCIF1-KO cells ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA, Dunnett’s test. ( D ) Colony formation assay detecting the colony ability of control and PCIF1-KO cells ( n = 3). *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( E and F ) The cell migration ( E ) and invasion ( F ) ability of control and PCIF1-KO cells was determined by Transwell assay ( n = 3). ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. Scale bar: 100 μm. ( G ) Flow cytometry assay for cell apoptosis in control and PCIF1-KO cells. Bottom: Representative images. Top: Quantification data ( n = 3). *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( H ) Cell cycle progression was detected by flow cytometric analyses in control and PCIF1-KO cells ( n = 3). ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: For PCIF1 overexpression plasmids, the full-length open reading frames of the human PCIF1 gene (NCBI Reference Sequence NM_022104.3) were cloned into the pLVX vector (GeneCopoeia).

Techniques: Western Blot, Expressing, Control, Cell Counting, CCK-8 Assay, Colony Assay, Comparison, Migration, Transwell Assay, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) The CTBP2 peptide identified in MS analysis after coimmunoprecipitation assay. ( B ) Immunoprecipitation and immunoblotting analysis of the interaction between CTBP2 and PCIF1. ( C ) GST pull-down assay of recombinant GST-CTBP2 and FLAG-PCIF1 proteins. The coprecipitated CTBP2 and PCIF1 proteins were detected by Western blot with anti-GST and anti-FLAG antibodies. ( D ) Strategy of CTBP2 variant proteins for mapping interaction domains with PCIF1. ( E and F ) Mapping of the binding domain of CTBP2 shows the potential binding site. The lysates were immunoprecipitated with anti-FLAG antibodies, followed by immunoblotting with anti-HA and anti-FLAG antibodies. Anti-IgG antibody was used as a negative control. ( G ) Analysis of colocalization of CTBP2 (green) with PCIF1 (red) by double immunofluorescence staining in control cells, PCIF1-KO cells, and CTBP2-KO cells. Nuclei are stained with DAPI (blue). Scale bar: 2 μm. ( H – J ) Pearson’s correlation analysis showed a positive correlation between CTBP2 and PCIF1 expression according to TCGA data ( H , n = 520), FAH-SYSU-Cohort1 ( I , n = 57), and HS-SYSU-Cohort2 ( J , n = 40). P value was calculated by Pearson’s correlation coefficient test. ( K ) Western blotting analyses detecting the CTBP2 expression in SCC25 control cells and CTBP2-KO cells. ( L and M ) LC-MS/MS quantification of the m 6 A/A ratio ( L ) and m 6 Am/A ratio ( M ) in mRNA obtained from HOK cells, control cells, PCIF1-KO cells, and CTBP2-KO cells ( n = 3). P > 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: For PCIF1 overexpression plasmids, the full-length open reading frames of the human PCIF1 gene (NCBI Reference Sequence NM_022104.3) were cloned into the pLVX vector (GeneCopoeia).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Pull Down Assay, Recombinant, Variant Assay, Binding Assay, Negative Control, Double Immunofluorescence Staining, Control, Staining, Expressing, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Venn diagram shows the integration of PCIF1-dependent modified genes and CTBP2-dependent modified genes; 382 genes are consistently modified by PCIF1 and CTBP2. ( B ) The top consensus m 6 Am motif identified in SCC25 cells with or without PCIF1 KO (left) and SCC25 cells with or without CTBP2 KO (right). ( C ) Bar plots showing the top 5 GO terms (Biological Process, DAVID) for PCIF1-dependent modified genes (top) and CTBP2-dependent modified genes (bottom). ( D ) Scatterplot of the translation ratios (TRs) in PCIF1-WT and PCIF1-KO SCC25 cells. TRs were calculated by division of the ribosome-binding transcript signals by input RNA-Seq signals. The PCIF1-WT SCC25 cell group served as the NC group. ( E ) Cumulative distribution plot of the translation efficiency (TE) distribution in cells with or without PCIF1 KO. ( F ) Bar plots showing the top 5 GO terms of genes with increased TRs. ( G ) Venn diagram shows the intersection of genes in GO Biological Process terms (regulation of transcription) from genes with increased TRs (left) and PCIF1- and CTBP2-dependent modified genes (right). ( H and I ) Representative images of PCIF1-dependent modified ( H ) and CTBP2-dependent modified ( I ) single m 6 Am sites on the transcripts of TET2. The 2 adenosine residues with a high score (red bars) were defined as m 6 Am sites.

Article Snippet: For PCIF1 overexpression plasmids, the full-length open reading frames of the human PCIF1 gene (NCBI Reference Sequence NM_022104.3) were cloned into the pLVX vector (GeneCopoeia).

Techniques: Modification, Binding Assay, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Real-time qPCR analysis of TET2 mRNA expression in control and PCIF1-KO cells ( n = 3). P > 0.05 by 1-way ANOVA with Tukey’s multiple-comparison test. ( B and C ) Real-time qPCR analysis of TET2 mRNA levels at the indicated times in control and PCIF1-KO SCC9 ( B ) and SCC25 ( C ) cells after actinomycin D treatment ( n = 3). P > 0.05 by 2-tailed unpaired Student’s t test. ( D and E ) TET2 expression ( D ) and DNA 5mC and 5hmC modification levels ( E ) were detected in control and PCIF1-KO cells. ( F and G ) TET2 expression ( F ) and DNA 5mC and 5hmC modification levels ( G ) were detected in SCC1 control cells and cells transfected with WT (OE) or mutant PCIF1 plasmid (OE mut ). MB, Methylene blue. ( H ) Schematic diagram of TET2 5′-UTR m 6 Am site mutations. Red arrows denote A594G and A604G mutation. ( I ) Luciferase activity of TET2 5′-UTR WT or TET2 5′-UTR m 6 Am site mutation (5′-UTR MUT) in SCC1 control cells and cells transfected with WT (OE) or mutant PCIF1 plasmid (OE mut ) ( n = 3). P > 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( J and K ) RNA immunoprecipitation (RIP)–qPCR analysis of TET2 mRNA retrieved by anti-PCIF1 ( J ) and anti-CTBP2 ( K ) antibody in control and PCIF1-KO cells ( n = 3). P > 0.05, *** P < 0.001 by 2-tailed unpaired Student’s t test. ( L ) RIP-qPCR analysis of TET2 mRNA retrieved by anti-PCIF1 antibody in CTBP2-KO cells transfected with vector (sgCTBP2), PCIF1 binding–defective mutant of CTBP2 (ΔCB-1), and WT CTBP2 ( n = 3). P > 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: For PCIF1 overexpression plasmids, the full-length open reading frames of the human PCIF1 gene (NCBI Reference Sequence NM_022104.3) were cloned into the pLVX vector (GeneCopoeia).

Techniques: Expressing, Control, Comparison, Modification, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, RNA Immunoprecipitation, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Experimental design of the carcinogen-induced HNSCC mouse model. Sac, sacrifice. ( B ) Diagram of Cre-dependent conditional knockout strategy for Pcif1. ( C ) Representative image of visible tongue lesions in the indicated groups. Scale bar: 1 mm. ( D ) Quantification of HNSCC lesion area in the indicated groups ( n = 8). * P < 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( E ) Representative H&E staining of HNSCC in the indicated groups. Scale bar: 100 μm. ( F ) Quantification of HNSCC number of lesions in the indicated groups ( n = 8). * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( G ) Quantification of HNSCC tumor grade in the indicated groups. * P < 0.05, ** P < 0.01 by Pearson’s χ 2 test. ( H ) Representative PCK staining of metastatic lymph node in the indicated groups. Scale bar: 300 μm. ( I ) Quantification of metastatic lymph node percentage in the indicated groups. * P < 0.05 by Pearson’s χ 2 test. ( J – L ) Representative dot blot image ( J ) and quantitative analysis of DNA 5mC ( K ) and 5hmC ( L ) modification levels in the indicated groups ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: For PCIF1 overexpression plasmids, the full-length open reading frames of the human PCIF1 gene (NCBI Reference Sequence NM_022104.3) were cloned into the pLVX vector (GeneCopoeia).

Techniques: Knock-Out, Comparison, Staining, Dot Blot, Modification

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Overview of the B AQR cryo-EM structure. Key subunits are colour coded. b , Compositional remodelling of the human spliceosome during catalytic activation. Subunits recruited or destabilized by the ATPase activities of PRP2 and Aquarius are indicated. Destabilized subunits can remain flexibly attached to the spliceosomes, often at lower stoichiometry. NTR, NTC-related proteins.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Cryo-EM Sample Prep, Activation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Overall maps of state A and B complexes depicted together with the final model. b , Local resolution of the state A maps, estimated in RELION and visualized in ChimeraX. c , Cryo-EM density snapshots. Various subunits are colored and labeled. d , Selected snapshots of modeled B AQR subunits are depicted together with the unsharpened map of the core region of the complex (map M2). e , Structural comparison between B act , B AQR , and C complexes. PRP2, Aquarius, SYF1, the U2 snRNA, and the pre-mRNA substrate are colored and labeled. Note the large-scale repositioning of the PRP2 RNA helicase during the conversion of B act to B AQR , and of the helicase Aquarius during the transition of B act /B AQR complexes to the C-stage spliceosome.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Domain composition and interactions of PRP2 in the B AQR complex. b , c , Structure and conformation of PRP2, the intron and SF3B1 in B AQR . Note that PRP2 NTD (the N-terminal domain) is not visible in c . d , In B AQR , PRP2 is moved in a cavity framed by PRP8 and SF3B1. The previous location and conformation of PRP2 and the intron in B act are shown in grey and black, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques:

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a–f , Interactions between PRP2, PRP8, SF3B1, CWC22 and SKIP. Interacting distances corresponding to polar and hydrophobic contacts are indicated as dashed lines. g , Overview of PRP2 and the composite molecular brake. PRP2’s conserved domains are color-coded. HB – helix-bundle; OB – oligonucleotide/oligosaccharide- binding fold; WH – winged-helix. h , Interactions between PPIL4, SKIP, and PRP2. Residues involved in contacts are depicted as spheres and shown in the same color as their interacting partner. i , Interactions between the wedge element of SKIP and PRP2 core . j , The wedge element of SKIP appears to lock the RecA domains of PRP2. The ADP molecule is modeled by superposition with Ct PRP2 (PDB 6zm2) and is shown for the sake of orientation. k , Superposition between human PRP2 in the open conformation (observed in B AQR ) and Ct PRP2 in the closed conformation (colored in teal) (PDB 6zm2). Equivalent residues of PRP2 interacting with SKIP in B AQR are shown for Ct PRP2. Structures from panels i and k are depicted in the same orientation. The red arrow indicates the movement of the RecA2-like domain during the helicase transition from the open to the closed conformation. l , Genetic interactions from yeast mapped on the structure of human B AQR . The cold-sensitive allele of Prp2p Q548N genetically interacts with the D450G and V502F substitutions of Hsh155p. Residues involved in genetic interactions in yeast (blue) are depicted as spheres and mapped on the B AQR model. m-o , Structural superposition of the human ( Hs ) and budding yeast ( Sc ) MA3 domain of CWC22, composed from HEAT repeats. The 10 th HEAT repeat (residues 454–491) of Cwc22p is required for the productive function of yeast Prp2p and interacts with the “hook” motif of PRP2 NTD and BUD13 in B AQR . This suggests that Prp2p, Cwc22p, and Bud13p may interact in a similar fashion in budding yeast spliceosomes, thus explaining the functional connection between Prp2p and Cwc22p. Note that human CWC22 residues that interact with PRP2 and BUD13 are conserved in budding yeast Cwc22p (shown in n and o). The protein subunits, U2 snRNA and the pre-mRNA substrate are colored and labeled.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Binding Assay, Functional Assay, Labeling

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a,b , Superposition between budding yeast (PDB 7DCO) and human (PDB 5Z56) B act complexes over equivalent residues from PHF5A. For clarity’s sake, only SF3B1, PRP2, the intron and U2 snRNA are depicted. All subunits are labeled accordingly. c , PRP2:RNA from human B AQR , as the only available structure of the human counterpart, was superimposed onto the Prp2p helicase from budding yeast B act and shown in the same orientation as in b. Note that the conformation of the RNA strand bound by human PRP2 or budding yeast Prp2p is virtually unchanged. d , Assignment of nucleotides bound by human PRP2 in B act based on the superposition. e , PRP2 translocates about 19 nucleotides towards the branch helix during the B act -to-B AQR transition. PRP2, RBMX2 and PPIL4 positions on the intron in different spliceosomal complexes are depicted. f , PRP2 translocation results in a substantial change in the intron’s conformation, dissociation of RBMX2 and recruitment of PPIL4 to the intron.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Labeling, Translocation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Relocation of PRP2 on the intron, RES complex dissociation from the translocated RNA and subsequent binding of PPIL4 to the latter. Pink, green and broken lines represent the PPT, the PPT region bound by SF3B1 within B act , and the PPT-equivalent region visible only within the budding yeast B act structure, respectively. b , Close-up view of the interfaces between PRP2 and other subunits in the B act complex. c , The equivalent view and orientation of the B AQR complex. d , SF3B1 exhibits primary and secondary hinged pockets for binding of BS-A and PPT, respectively. SF3B1 adopts a loose conformation in B AQR , whereby the primary and secondary pockets are occupied and free, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Binding Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Destabilization of SF3B6, de-structuring of SF3B1 NTD , and the reorientation of PRP8’s EN (endonuclease-like, PRP8 EN ) and RH (RNase H-like, PRP8 RH ) domains upon PRP2 translocation. PRP2 is located on the plane above SF3B1 HEAT and is not shown for clarity’s sake. The reactive BS-A, the guanosine of the 5’SS, and the catalytic metal ions are shown as spheres and labeled. b , Close-up view of SF3B1, the intron, and the U2 snRNA from B AQR (color-coded) and B act (grey and black) after superposition of SF3B1’s equivalent residues. c , The binding pocket of the BS-A (primary pocket) is virtually unchanged in B act and B AQR . d , Cycle of SF3B1 transitions in splicing. The conformational transitions of SF3B1 are depicted based on cryo-EM structures and biochemical analyses. Here we refer to the intermediates II and III as pre-A1 and pre-A2 for clarity. The key spliceosome complexes representative for the SF3B1 intermediates shown here are: 17S U2 snRNP , , pre-A1 (A-like cross-exon complex bound by spliceostatin A ), pre-A2 (ref. ), A-to-B act (reviewed in refs. , , ), B AQR (this work), the SF3B complex , . Helicases that facilitate the transitions are shown in red. The helicase DHX15, colored magenta, mediates the disassembly of kinetically-slowed complexes (e.g. formed on suboptimal introns, weak splice sites and PPTs, multiple branch sites or cryptic sites – ).

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Translocation Assay, Labeling, Binding Assay, Cryo-EM Sample Prep

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Interactions between the subunits of the molecular brake. Interacting residues are shown as spheres depicted in the same colour as their binding partner. The domains of PRP2 RecA1, RecA2, HB and NTD are depicted. The anchoring surfaces of the brake elements to PRP8 (grey oval) are indicated. The inset shows a schematic representation of the molecular brake, in which the subunits are coloured as in the surface and cartoon representation. CWC15 is yellow. b , Interactions of the intron with PRP2 NTD , PPIL4 and SKIP. c , Interactions of PPIL4 with CWC15 and SKIP. d , e , The wedge element of SKIP intercalates between RecA1, RecA2 and the HB domains of PRP2, indicating inhibition of translocation. PPIL4, SKIP and CWC15 are red, cyan and yellow, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Binding Assay, Inhibition, Translocation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Size-exclusion chromatography (SEC) profiles of PRP2 (137-1022), PPIL4 and the mixture of the two proteins shows that PRP2 (137-1022) forms a stable complex with PPIL4, in an RNA-independent manner. PRP2 (137-1022) comprises both the modeled PRP2 NTD ( i.e ., the pin, clip, and hook elements) and the helicase core. b , SDS-PAGE gels corresponding to the fractions from a . The in vitro reconstitution experiments were repeated two times, using two independent preparations of PRP2 (137–1022) and PPIL4. c , SDS-PAGE gel of SEC fractions showing the reconstitution of a stable PRP2-PPIL4 complex in vivo by co-expression in insect cells. PRP2 (137–1022) and PPIL4 were co-expressed in Sf9 insect cells from individual baculoviruses. The complex was captured by Strep-Tactin affinity followed by SEC. The preparation was performed twice with similar results d , The RNA-binding activity of human PRP2 in the presence of GPKOW and PPIL4. The ability of PRP2 (137–1022) and of the purified PRP2 (137–1022)-PPIL4 and PRP2 (137-1022)-GPKOW complexes to bind a Cyanine 5 (Cy5)-labeled RNA substrate was evidenced by EMSA. The RNA substrate comprised an RNA duplex, followed by 30 nucleotides 3′ single-stranded overhang, mimicking PRP2’s spliceosome substrate observed in B AQR . The free RNA substrate was separated from PRP2-bound (or cofactor-bound) species on a 5% polyacrylamide native gel. The EMSA gels were imaged at the Cy5 excitation peak. The assays were repeated two times. e , The RNA binding activity of human PPIL4 in the absence and presence of PRP2 (137–1022). Compared to the EMSA shown in d , PPIL4 was added in a 5-fold excess over PRP2 (137–1022) and PRP2’s final assay concentrations are indicated above the last three gels lanes. Note the apparent increase in PRP2’s RNA affinity in the presence of PPIL4. The EMSAs were repeated three times. f , SEC profiles showing the formation of a stable complex between PRP2 (137–1022) and GPKOW, in an RNA-independent manner. The SDS-PAGE corresponding to the complex purified by SEC is shown. g , The RNA-binding activity of human GPKOW assessed by an EMSA. The same RNA substrate was used as in d and the assay was repeated four times. h , The helicase activity of human PRP2 was investigated using a fluorescence-based assay. Compared to the gel-based helicase assays shown in i-l , the fluorescence-based assay employs a dual-labeled helicase substrate. Displacement of the labeled strand by the helicase leads to the formation of an intramolecular hairpin, which brings in proximity the Cy5 fluorophore and its spectrally overlapping quencher (BHQ-2). The decrease in substrate’s fluorescence upon unwinding is monitored as a function of time. Several representative fluorescence traces of PRP2, recorded under different experimental conditions and in the presence/absence of helicase cofactors, are shown together with the unwinding curves of Prp22p, used as a positive control. i , The helicase/unwinding activity of human PRP2 and of the PRP2-GPKOW complex. To assess the ability of PRP2 (137–1022) and of the purified, in vitro reconstituted PRP2 (137–1022)-GPKOW complex to unwind RNA-RNA duplexes, the purified protein samples were mixed with the Cy5-labeled helicase substrate (depicted on the right with the labeled strand colored in red) in the presence (or absence) of ATP and of a competitor DNA (green). Following a 1-hour incubation, the samples were analyzed on a native 14% polyacrylamide gel and imaged by in-gel fluorescence. Budding yeast Prp22p was used as a positive control. All gel-based helicase assays were repeated at least two times. j , The helicase activity of the in vivo reconstituted PRP2 (137–1022)-PPIL4 complex, in the absence or presence of GPKOW. k , The helicase activity of human PRP2 (137–1022) in the presence or absence of its cofactor GPKOW. Compared to the assay shown in i , the GPKOW cofactor was added to the purified helicase in a 5-fold excess. The concentrations indicated above the native gel represent the final assay concentrations of PRP2 (137–1022) or Prp22p. The RNA bands labeled with an asterisk represent, most likely, degradation products. l , Comparative helicase activities of the PRP2 (137–1022)-PPIL4 and PRP2 (137–1022)-GPKOW complexes. For gel source data, see Supplementary Figs. and .

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Size-exclusion Chromatography, SDS Page, In Vitro, In Vivo, Expressing, RNA Binding Assay, Activity Assay, Purification, Labeling, Fluorescence, Positive Control, Incubation

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , During the B act (PDB 5Z57) to the B AQR transition, translocation of PRP2 in a 3′-to-5′ direction results in the destabilization of the RES complex (RBMX2, BUD13, SNIP1). In addition, SRRM1 and SF3B6/p14 are no longer observed in B AQR due to PRP2-induced SF3B1 opening. During the remodeling of B AQR and transition to the C complex (PDB 5yzg, PDB 6zym), the remaining SF3B/SF3A subunits and CWC24 are released from the branch helix and the 5’SS. The branch helix then moves to the catalytic center, bringing the BS-A and the 5’SS GU nucleotides in proximity for the branching reaction. The different human spliceosome states were structurally aligned by using the PRP8 subunit as a reference and are depicted in two different orientations. The spliceosome subunits are color-coded and shown in cartoon representation. Spliceosome subunits not undergoing significant rearrangement were omitted for the sake of simplicity. b , Repositioning of the branch helix (U2/BS) during the transition from B act to B AQR and then to C complexes. The different spliceosome states, B act (PDB 5Z57), B AQR (this work), and C complex (PDB 5yzg, PDB 6zym), were aligned using the PRP8 subunit as a reference. All protein subunits, except PRP8, were omitted and the RNA moieties were color-coded. The reactive BS-A and the 5’SS GU nucleotides are shown as spheres and colored red and light green, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Translocation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Catalytic activation depicted as structural transitions between B act , B AQR and B*. B* and the post-branching C complex have similar structures. Most subunits are shown as surface representations. SYF1 and SYF3 are shown as ribbons to enable better visualization of Aquarius. b , Structure-based model of human spliceosome remodelling by PRP2 and Aquarius. The hypothetical intermediate between B act and B AQR considers the following: (1) the molecular brake can form only after the binding of PPIL4 to the RNA, the binding site of which becomes available after dissociation of the RES complex by the translocation of PRP2; and (2) the advance of PRP2 should start stripping the intron from SF3B1 and promote the loose conformation of SF3B1. The hypothetical intermediate between B AQR and B* was generated by considering that Aquarius should induce complete displacement of the branch duplex from SF3B1, liberating BS-A from its binding pocket. Consequently, SF3B1 will transit from the loose to the open conformation. Because the HEAT repeats that bind PRP8 (in B AQR ) rearrange after loose-to-open conformation, SF3B1 dissociates from PRP8. This likely causes the complete destabilization of the SF3A–SF3B complexes and PRP2 from the spliceosome. c , Model of the spliceosome remodelling by Prp2p in budding yeast (S. cerevisiae) , based on the similarities and differences with the human counterpart.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Activation Assay, Binding Assay, Translocation Assay, Stripping Membranes, Generated

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Lines of structural communication between Aquarius and the branch duplex in B AQR . Aquarius and PRP2 are in diametrically-opposed locations of the B AQR spliceosome. A continuous bridge of proteins is present between Aquarius and the SF3B1:branch duplex. Most of these proteins are SF3A/B subunits and PPIE. Another bridge that primarily involves the RBM22 protein is between Aquarius and U6 catalytic core. The intron region not visible in the density is dashed. Aquarius is depicted in red (RecA-like domains) and light blue (accessory domains). The first nucleotide of the intron (G +1 ) is positioned in the catalytic center. Other subunits of the spliceosome, including SYF1 and ISY1, are not shown for the sake of clarity b , Subunits of the C complex (pdb 5yzg) are shown in the same orientation as in a . The U6 snRNA was used as a reference for the superposition between the C and B AQR complexes. Note that PRP2 and the SF3A/B complex have dissociated and the BS-A has been relocated to the catalytic centre (see also c , below). RBM22’s orientation has remained virtually the same in B AQR and C complexes. The subunits are colored as in a and labeled. The B AQR and C complexes were superimposed over PRP8 (not shown) and U6 snRNA c , The BS-A juxtaposed to the first nucleotide of the intron in the catalytic center of the C complex. The catalytic metal ions are shown as magenta spheres. d , Superposition between the “open” (observed in the apo SF3B complex, PDB 5IFE) and the “loose” states (observed in B AQR ) of SF3B1. Except for SF3B1 OPEN , all shown subunits belong to the B AQR complex. e , B AQR and the SF3B complex in its apo form (PDB 5IFE) were superimposed over equivalent residues from PHF5A (not shown in the figures d, f and g for clarity’s sake). Note that the HEAT repeats H16-H20 of SF3B1 adopt virtually identical conformations in the “loose” and “open” states, while the other helical repeats are substantially reorganized. f–g , The BS-A’s release from the primary pocket of SF3B1 likely induces rearrangement of the HEAT repeats upon the “loose” to “open” conformational transition. Consequently, the contacts between HEAT repeats and PRP8, and those between SF3B2 (attached to the HEAT repeats) and the U6 snRNA might get disrupted, causing the complete dissociation of the SF3A/B complexes from the spliceosome.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Labeling