Journal: Molecular Cancer

Article Title: CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

doi: 10.1186/s12943-021-01481-w

Figure Lengend Snippet: circRTN4 stabilizes RAB11FIP1 by preventing its ubiquitination and degradation. A Heat-map showing 99 circRTN4-interacting proteins in PDAC. Biotin-labelled circRTN4, RTN4 mRNA, circGFP were used to pull down circRTN4-interacting proteins. Mass spectrometry analysis was performed to identify the interacting proteins. B CircRTN4 interacted with RAB11FIP1 in PDAC cells, as revealed by RIP assay. C Bioinformatics analysis by PRIdictor revealed the RAB11FIP1-binding site (The seed region of interaction was in red) on circRTN4. Mutating RAB11FIP1-binding site on circRTN4 (The mutated seed region of interaction was in green) inhibited circRTN4-RAB11FIP1 interaction in PANC-1 cells, as revealed by circRNA pulldown assay. D CircRTN4 knockdown inhibited RAB11FIP1 expression in mice xenograft. E CircRTN4 knockdown did not affect RAB11FIP1 mRNA level in PDAC cells. F CircRTN4 knockdown decreased the stability of RAB11FIP1 after inhibition of protein synthesis by cycloheximide in PANC-1 cells. G 3-Dimensional structure of the circRTN4-RAB11FIP1 interaction revealed that circRTN4 blocked the ubiquitination site Lys578 of RAB11FIP1. H Immunoprecipitation with anti-RAB11FIP1 antibody in PANC-1 cells after circRTN4 knockdown, followed by immunoblotting analysis with anti-ubiquitin or anti-RAB11FIP1 antibody. CircRTN4 knockdown increased ubiquitination of RAB11FIP1. I RAB11FIP1 expression were upregulated in PDAC primary tumors and was positively correlated with circRTN4 level. Data represent mean ± SD from at least three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Briefly, cells were incubated magnetic beads labelled with antibody against RAB11FIP1 (rabbit; 16,778–1-AP Proteintech) or TWF1 (mouse; sc-376,539 Santa Cruz Biotechnology, TX, USA) overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Binding Assay, Knockdown, Expressing, Inhibition, Immunoprecipitation, Western Blot

Journal: Molecular Cancer

Article Title: CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

doi: 10.1186/s12943-021-01481-w

Figure Lengend Snippet: circRTN4 stabilizes RAB11FIP1 to promote EMT in PDAC. A N-cadherin expression was inhibited after circRTN4 knockdown in PANC-1 cells. B CircRTN4 knockdown in mice subcutaneous tumors inhibited N-cadherin expression. C Expression of EMT markers Slug, Snai1, Twist and Zeb1 were reduced after circRTN4 knockdown in PDAC cells. D Expression of Slug, Snai1, Twist and Zeb1 in circRTN4-overexpressing HPDE cells were rescued after RAB11FIP1 knockdown. E Slug, Snai1, Twist and Zeb1 expression were upregulated in PDAC primary tumors and were positively correlated with circRTN4 level. Data represent mean ± SD from at least three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Briefly, cells were incubated magnetic beads labelled with antibody against RAB11FIP1 (rabbit; 16,778–1-AP Proteintech) or TWF1 (mouse; sc-376,539 Santa Cruz Biotechnology, TX, USA) overnight at 4 °C.

Techniques: Expressing, Knockdown

Journal: Molecular cell

Article Title: A complex of U1 snRNP with cleavage and polyadenylation factors controls telescripting, regulating mRNA transcription in human cells

doi: 10.1016/j.molcel.2019.08.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Fip1 , Bethyl laboratories , A301–462A.

Techniques: Recombinant, RNA Sequencing, Control, Software

Journal: Nature Communications

Article Title: The missense mutation Y65C in PQBP1 causes microcephaly and cognitive deficits through a combination of partial loss-of-function and gain-of-function effects

doi: 10.1038/s41467-025-68202-5

Figure Lengend Snippet: a Venn diagram delineating the overlapping distribution of differential binding proteins across parallel IP-MS datasets. n = 3 mice in each group in each independent experiment. b Top 10 enriched biological processes ranked by P -value. Analysis of the overlapping proteins from ( a ) using a one-sided hypergeometric test with Benjamini–Hochberg FDR correction. Numbers and the size of circles represent counts. c Heatmap visualization reveals differential expression patterns of enhanced binding proteins from ( a ). IBAQ intensities from duplicate IP-MS experiments were quantile-normalized and averaged. d Scatter plot depicting the genes with APA changes (FDR < 0.05) in E12.5 Pqbp1 Y65C/Y cortex. Blue: Transcripts with proximal PAS preference in Y65C. Red: Transcripts demonstrating distal PAS preference in Y65C. n = 3 mice in each group. e The genes with APA changes (FDR < 0.05) in E12.5 Pqbp1-cKO cortex. Blue: transcripts with proximal PAS preference in cKO compared to the control; Red: Transcripts demonstrating distal PAS preference in cKO. n = 3 mice in each group. f Venn diagram shows the overlapped targets between Pqbp1 Y65C/Y and Pqbp1-cKO . g The genes overlaping with known ESC self-renewal regulators. h Integrative Genomics Viewer tracks show the representative gene reads of Marcksl1 from RNA-seq. i qRT-PCR measures the ratios of transcripts with the extended 3′ UTR of Marcksl1 . The locations of primer sets used to amplify specific regions are marked (top). n = 3 mice in each group, p = 0.0329. j CoIP of PQBP1 and FIP1L1. The cortex of mice at E15.5 was immunoprecipitated with anti-PQBP1 antibodies. n = 3 biologically independent experiments, p = 0.0085. k , l Dual-luciferase reporter assays for Flag, Flag-PQBP1, and Flag-PQBP1 Y65C groups. The ratios of R/F (Renilla luciferase 480 nm/firefly luciferase 560 nm) were normalized to the Flag group. CMV, CMV promoter; R luc, Renilla luciferase gene; IRES, internal ribosomal entry site; F luc, firefly luciferase gene. n = 5 biologically independent experiments, Flag vs Flag PQBP1: p < 0.0001; Flag vs Flag PQBP1 Y65C : p = 0.0002. All quantification data are represented as mean ± SD. j Two-tailed unpaired Student’s t test. l Two-tailed one-way ANOVA with Tukey’s multiple comparisons test to adjust for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were PQBP1 (16264-1-AP, Proteintech, 1:1000 for use); RPS3 (ab128995, abcam, 1:1000 for use); RPL13 (ab134961, abcam, 1:1000 for use); FIP1L1 (83863-2-RR, Proteintech, 1:1000 for use); anti-α-tubulin (T9026, Sigma-Aldrich, 1:5000 for use); anti-actin (T0022, Affinity, 1:5000 for use).

Techniques: Binding Assay, Protein-Protein interactions, Quantitative Proteomics, Control, RNA Sequencing, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Two Tailed Test

Journal: Molecular cell

Article Title: Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation

doi: 10.1016/j.molcel.2017.11.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Fip1-RD peptide , GenScript , Custom synthesis: SC1208/U2711BI160_1.

Techniques: Recombinant, Reporter Assay, Transfection, Cloning, In Vitro, Software