Journal: Nature Communications

Article Title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs

doi: 10.1038/s41467-025-64848-3

Figure Lengend Snippet: a Experimental and analytical workflow of adopting different PB proteins for STAMP and HyperTRIBE. b Immunofluorescence showing subcellular localization of APOBEC1, ADAR2dd, APOBEC1-DDX6, and LSM14A-ADAR2dd proteins with PB marker EDC4 protein, respectively. The experiment was repeated independently for 2 times with similar results. c - c’ Metaplots showing the sequencing depth-normalized distribution of the number of confident edit sites (≥0.65 confidence score) detected in cells expressing PB fusion proteins with high editing efficiency or deaminase alone. c C-to-U edit sites. c’ A-to-I edit sites. d - d’ Volcano plots comparing the editing score (ES) between cells expressing PB fusion protein and those expressing only deaminase. X and Y axes represent log2 fold change (LFC) and −log10( p value), respectively. Red dots represent the target mRNAs passing the respective threshold. Statistical significance was calculated using two-sided Fisher’s exact test followed by Benjamini–Hochberg correction. d APOBEC1-DDX6 vs. APOBEC1. d’ LSM14A-ADAR2dd vs. ADAR2dd. e Venn diagram showing the overlap between target mRNAs identified by APOBEC1-DDX6 and LSM14A-ADAR2dd, respectively. Statistical significance was calculated using the one-sided hypergeometric test. Source data are provided in the Source Data file.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP, 1:500), EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382, 1:50), EDC4 rabbit polyclonal antibody (Proteintech, 17737-1-AP, 1:500), MYC tag mouse monoclonal antibody (Proteintech, 66004-1-Ig, 1:1000), HA tag rat monoclonal antibody (Roche, 11867423001, 1:1000), and FLAG tag mouse monoclonal antibody (Proteintech, 66008-4-Ig, 1:1000).

Techniques: Immunofluorescence, Marker, Sequencing, Expressing

Journal: Nature Communications

Article Title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs

doi: 10.1038/s41467-025-64848-3

Figure Lengend Snippet: a Immunofluorescence showing localization of APOBEC1, ADAR2dd, and PB marker EDC4 proteins in control HCT116 cells and co-localization of APOBEC1-DDX6 and LSM14A-ADAR2dd proteins with EDC4 proteins in dual-editing HCT116 cells. The experiment was repeated independently for 2 times with similar results. b - b’ Scatter plots showing the correlation of ES between single-editing and dual-editing HCT116 cells. The Pearson correlation coefficient R and p value (two-sided) are indicated in the top left corner. b C-to-U edits. b’ A-to-I edits. c Venn diagram showing the overlap between the target mRNAs identified by APOBEC1-DDX6 and LSM14A-ADAR2dd in dual-editing HCT116 cells. Statistical significance was calculated using the one-sided hypergeometric test. d Venn diagram showing the overlap between the target mRNAs identified by APOBEC1-DDX6 and LSM14A-ADAR2dd in dual-editing HEK293T cells. Statistical significance was calculated using the one-sided hypergeometric test. e Venn diagram showing the overlap between the LSM14A-DDX6-associated mRNAs identified in dual-editing HCT116 and HEK293T cells. Statistical significance was calculated using the one-sided hypergeometric test. f Gene ontology analysis of LSM14A-DDX6-associated mRNAs shared between the two cell lines. Statistical significance was calculated using the clusterProfiler R package with default settings. g Venn diagram showing the overlap between PB-associated mRNAs identified by LSM14A-FAPS, DDX6-eCLIP-seq, and LSM14A-DDX6-associated mRNAs identified by PB-TRIBE-STAMP in HEK293T cells. h - h’ RIP-RT-qPCR validation of LSM14A-DDX6-associated mRNAs in HCT116 cells. Different groups of genes, identified by other methods or exclusively by PB-TRIBE-STAMP are indicated with different colors. Three independent biological replicates were analyzed. h Immunoprecipitation using DDX6 antibody. h’ Immunoprecipitation using EDC4 antibody. Error bars represent mean ± SD. Statistical significance was calculated using ordinary one-way ANOVA (default in Prism 9) (ns: p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). i - i” RNAscope confocal images showing co-localization of XBP1 mRNAs ( i ), SREBF2 mRNAs ( i’ ), and HMGA2 mRNAs ( I” ) with EDC4 proteins. Source data are provided in the Source Data file.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP, 1:500), EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382, 1:50), EDC4 rabbit polyclonal antibody (Proteintech, 17737-1-AP, 1:500), MYC tag mouse monoclonal antibody (Proteintech, 66004-1-Ig, 1:1000), HA tag rat monoclonal antibody (Roche, 11867423001, 1:1000), and FLAG tag mouse monoclonal antibody (Proteintech, 66008-4-Ig, 1:1000).

Techniques: Immunofluorescence, Marker, Control, Quantitative RT-PCR, Biomarker Discovery, Immunoprecipitation, RNAscope