AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma. (2022)
Figure 1

Figure 1: Che-1 interacts with paraspeckle components.

A: Experimental and computational workflow followed to test the relationship between Che-1 and paraspeckle components. (Left) Mass spectrometry analysis was performed to identify Che-1 interactome followed by: (Middle) Che-1 SFPQ/NONO co-immunoprecipitation (Co-IP) immunofluorescence (IF) and Proximity ligation assay (PLA) to evaluate physical interaction. (Right) Then eCLIP data of 356 RNA Binding Protein in K562 cell line was evaluated at NEAT1 locus followed by Che-1 RNA immunoprecipitation (RIP) and co-localization in KMS27 and RPMI8226 Multiple Myeloma (MM) cell lines.

B: Scatter Plot of mass spectrometry assay. Dots depict protein intensity (IBAQ) and relative significance obtained from mass spectrometry assay. X-axis: Difference of Che-1 intensity versus IP control; on the y-axis are the corresponding −log10 P-values. Significant Che-1 interacting proteins relevant in this study are represented in blue. For a complete list of all significant Che-1 interactors, please refer to Dataset EV1.

C: Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies (abs) and analyzed by western blot (WB) with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.

D: (Top) qRT-PCR approach described in the schematic representation. (Bottom) RIP-qPCR analysis with anti-Che-1 specific antibody or control rabbit IgG of Kms27 MM cells. Data are expressed as percent of Input. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ***P<0.005, ****P<0.001, n.s. = not significant).

E: Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization (FISH) staining of NEAT1 (Stellaris) with immunofluorescence of Che-1. Scale bar 10 μm.

F: (Left) RT-qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ****P<0.001). (Right) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs. Figure 2: Che-1 and NEAT1 colocalize on the DNA.

A: Experimental and computational workflows developed to identify NEAT1/Che-1 co-binding sites in MM Kms27 cells. ChIRP-seq and ChIP-seq were used to identify NEAT1 and Che-1 biding sites, respectively. Then, normalized read enrichment of both experiments was assessed at 92,226 transcription starting sites (coordinates from Ensembl GRCh37). Peaks were classified using an iterative approach of selection and assignment based on Jaccard scoring (Middle). Finally, peaks were classified into only NEAT1, only Che, Che-1/NEAT1 co-binding.

B: Genomic annotation of significant peaks from the NEAT1 ChIRP-seq (Top bar) and Che-1 ChIP-seq (Bottom bar) experiments. Peaks were classified among six categories: Non-coding, Exon, Intron, Promoter-TTS, TTS, UTR according to Homer annotation tool. Each category is shown with a different color. The percentage of classified peaks for each category is shown as experiment-specific percentage (x-axis).

C: Bar diagram showing the number of peaks shared between NEAT1/Che-1 (black), Che-1 only, (red), NEAT1 only (yellow). The peak number is shown at the top of each bar.

D: Bar diagram showing the relative distance of each Che-1/NEAT1 co-binding peak to the closest gene. Distance is calculated as absolute value and is expressed in kilobase. The number of peaks per category at the top of each bar. Bar height represents the proportion of peaks included in each category to the total (N=14654).

E: Gene Ontology (Left) and KEGG enrichment pathways analysis (Right) of Che-1/NEAT1 co-binding sites. The top 6 enriched terms are shown. Only ontology terms significant by both the binomial and hypergeometric tests using the multiple hypothesis correction false discovery rate are considered in this analysis. The represented q-value (x-axis) corresponds to the hypergeometric test.

F: Che-1 ChIP-seq, NEAT1 ChIRP, H3K27ac ChIP-seq, Che-1 only, NEAT1 only, Co-Binding sites, ENSEMBLE TSS annotation (From Top to Bottom) signals at selected genomic loci (Genome reference: GRCh37). Color scheme: RED: Che-1 only signals; Yellow: NEAT1 only signals; Dark Green: co-binding sites.