Journal: Nature Communications

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA

doi: 10.1038/s41467-022-30323-6

Figure Lengend Snippet: a Illustration of experimental approach tracing the effect of developmental loss of subunit POLR3G, subunit-specific disruption of POLR3G, and cancer-associated re-establishment of POLR3G and accompanying chromatin features, which collectively identify POLR3G-driven modulation of Pol III transcription potential in proliferating cells. b Pol III subunit and transcription factor legend corresponding to genome-wide maps in panels c – k , including POLR3A/RPC1 (3A); POLR3B/RPC2 (3B); POLR1D/RPAC2 (1D); POLR3C/RPC3 (3C); POLR3G/RPC7α (3G); POLR3GL/RPC7β (3GL); POLR3D/RPC4 (3D); POLR3E/RPC5 (3E); BRF1/TFIIIB90; TF3C1/TFIIIC220. Corresponding subcomplex indicated. c – k Example ChIP-seq read signals for each subunit/factor are shown in THP-1 monocytes across canonical Pol III-transcribed genes of varying promoter architecture, including ribosomal 5 S rRNA genes (type 1 promoter; panel c ), tRNA, 7SL, vault, and snaR-A ncRNA genes (type 2 promoter, panels d – g , respectively), and Y, U6, 7SK, and RMRP ncRNA genes (type 3 promoter, panels h – k , respectively). Gene labels include Unique RNA Sequence (URS) identifiers assigned by (and connected to) the RNAcentral database. Bottom panel illustrates corresponding promoter architecture classification. l Cartoon schematic of the Pol III protein complex with emphasis on subunits mapped in this study (labeled) and the corresponding color code for genomic signal plots. Illustration serves as general reference guide inspired by previous structural reconstructions (Hoffmann et al., Nature 2015) . m Visualization of the correlation between log2(normalized read densities) for paralogous Pol III subunits, POLR3G and POLR3GL, at Pol III complex–occupied genes in THP-1 monocytes. Source Data are provided as a Source Data file.

Article Snippet: THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology).

Techniques: Disruption, Genome Wide, ChIP-sequencing, Sequencing, Labeling

Journal: Nature Communications

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA

doi: 10.1038/s41467-022-30323-6

Figure Lengend Snippet: a RT-qPCR analysis of POLR3G and POLR3GL mRNA, and snaR-A ncRNA levels following THP-1 differentiation ( n = 3 biologically independent experiments). P -value = 0.00088 ( POLR3G ), 0.099 ( POLR3GL ), 0.00014 (snaR-A seq1), 0.0013 (snaR-A seq2). Significance calculated using Student two-sided t -test. b Immunoblots for Pol III subunits mapped in THP-1 cells, ±72-h PMA-induced differentiation. *Lamin B2 corresponds to POLR3A and POLR3GL. Observations representative of 2 or more independent experiments. c , d Volcano plot visualization of dynamic POLR3G ( c ) and POLR3GL ( d ) genomic occupancy ± 72-h PMA-induced differentiation. Gene class indicated by color legend. Significance calculated using edgeR two-sided exactTest function, Benjamini–Hochberg corrected p -value. e Heatmap visualization of the log2(fold change) of Pol III subunit occupancy for POLR3B, POLR1D, POLR3G, POLR3GL, and POLR3D in THP-1 cells ± 72-h PMA treatment. Heatmap is ordered by median fold change across all subunits. Inset highlights top 35 (10%) of ranked genes. f ChIP-seq track visualization for POLR3B, POLR1D, POLR3G, POLR3GL, and POLR3D in THP-1 cells before (left) and after 72-h PMA treatment (right) across a subset of SNAR-A genes encoding snaR-A ncRNA. g Heatmap visualization of dynamic small RNA profiles in THP-1 monocytes, including nascent, steady-state, nuclear, cytoplasmic, and exosomal small RNA fractions. All heatmaps are ordered by the level of nascent RNA abundance in THP-1 monocytes. Corresponding genes for all heatmaps indicated by color legend on right. h RT-qPCR analysis of POLR3G and POLR3GL mRNA, and snaR-A ncRNA levels following THP-1 differentiation and lentiviral transduction with control or POLR3G-specific CRISPR activation particles (sc-437282 and sc-417072-LAC-2) ( n = 6 biologically independent experiments). P -value = 0.00012 ( POLR3G ), 0.00046 ( POLR3GL ), 5.79e-7 (snaR-A seq1), 4.77e-5 (snaR-A seq2). Significance calculated using Student two-sided t -test. Data are presented as mean ± standard error mean (SEM) from the indicated number of independent samples ( a , h ). Molecular weights are indicated in kDa. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns, nonsignificant. Source Data are provided as a Source Data file.

Article Snippet: THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, ChIP-sequencing, Transduction, Control, CRISPR, Activation Assay

Journal: Nature Communications

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA

doi: 10.1038/s41467-022-30323-6

Figure Lengend Snippet: a Pol III subunit gene expression profiles across hematopoietic progenitor cells and distinct differentiation immune cell lineages, including myeloid, B cell, natural killer (NK), CD4 + T, CD8 + T, and γδ T cells, with emphasis on POLR3G and POLR3GL . b Pol III-transcribed gene accessibility (ATAC-seq) across hematopoietic progenitor cells and distinct differentiation immune cell lineages corresponding to Fig. 3a. Individual rows (genes) are ranked by median accessibility score across cell types. Loss of gene accessibility is signified by weakened ATAC-seq signal across a subset of genes in differentiated immune cell populations. c Heatmap visualization of correlations between gene accessibility and gene expression ratio of POLR3G:POLR3GL . Correlation represents integration of average context-specific expression level (Fig. 3a) and average context-specific gene accessibility (Fig. 3b). Corresponding genes indicated by color legend on right. d – f Individual correlation profiles of SNAR-A ( d ), BCYRN1 ( e ), and tRNA-Ala gene (URS0000209048) chromatin accessibility with POLR3G:POLR3GL gene expression ratios in primary immune cells depicted in heatmap (Fig. 3c). Pearson’s correlation, corresponding p -values computed as two-sided test. g POLR3G gene expression profile in primary immune CD4 + T cells before and after co-stimulation with anti-CD3/CD28 Dynabeads . From left to right, N = 37,40 independent experiments. Statistical analysis with a two-sided Wilcoxon rank-sum test. P = 2.3e-11. h POLR3GL gene expression profile in primary immune CD4 + T cells before and after stimulation. From left to right, N = 37, 40 independent experiments. Statistical analysis with a two-sided Wilcoxon rank-sum test. P = 0.0047. i SNAR-A gene accessibility profile in primary immune CD4 + T cells before and after stimulation. From left to right, N = 40,37 independent experiments. Statistical analysis with a two-sided Wilcoxon rank-sum test. P = 2.56e-5. Box plot center lines correspond to median, lower and upper hinges first and third quartile. Whiskers present minimum and maximum values not exceeding 1.5*IQR beyond first and third quartile. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns, nonsignificant. Source Data are provided as a Source Data file.

Article Snippet: THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology).

Techniques: Gene Expression, Expressing

Journal: Nature Communications

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA

doi: 10.1038/s41467-022-30323-6

Figure Lengend Snippet: a Diagram of TCGA patient-matched normal and primary solid tumor cancer types profiled for POLR3G and POLR3GL gene expression levels in this study. b – j Gene expression ratios of POLR3G/POLR3GL in lung squamous cell carcinoma ( b , n = 51), lung adenocarcinoma ( c , n = 58), stomach adenocarcinoma ( d , n = 32), esophageal carcinoma ( e , n = 11), kidney chromophobe ( f , n = 25), cholangiocarcinoma ( g , n = 9), colorectal adenocarcinoma ( h , n = 32), bladder urothelial carcinoma ( i , n = 19), and uterine corpus endometrial carcinoma ( j , n = 10). Gray arrows represent individual patient-matched normal and primary solid tumors. Corresponding p -values determined using paired Wilcoxon signed-rank two-sided test. Box plot center lines correspond to median, lower and upper hinges first and third quartile. Whiskers present minimum and maximum values not exceeding 1.5*IQR beyond first and third quartile. k 3D scatterplot visualization of the subrepertoire of Pol III-transcribed genes with correlative POLR3G and predicted gene activity in The Cancer Genome Atlas (TCGA) and primary immune profiling experiments. l – n Kaplan–Meier analysis of overall survival of TCGA donors stratified by high POLR3G gene expression (top tertile; top panels) or high POLR3GL gene expression (top tertile; bottom panels) in lung adenocarcinoma ( l ), esophageal carcinoma ( m ), and bladder urothelial carcinoma ( n ). Corresponding p -values determined using log-rank two-sided test; HR = hazard ratio risk of dying. Source Data are provided as a Source Data file.

Article Snippet: THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology).

Techniques: Gene Expression, Activity Assay

Journal: Nature Communications

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA

doi: 10.1038/s41467-022-30323-6

Figure Lengend Snippet: a THP-1 growth curves were generated using MTT cell proliferation assays in cells exposed to 0 (control), 25, 50, 75, and 100 uM ML-60218. b , c Volcano plot visualization of dynamic POLR3G ( b ) and POLR3GL ( c ) genomic occupancy in THP-1 cells ± 4 h exposure to Pol III inhibitor ML-60218 (25 uM). Gene class indicated by color legend. Significance calculated using edgeR two-sided exactTest function, Benjamini–Hochberg corrected p -value. d Heatmap of log2(fold change) in small RNA abundance for Pol III-transcribed genes in THP-1 cells at 0, 1, 2, 3, and 4 h post-exposure to Pol III inhibitor ML-60218 (25 uM). Heatmap is ordered by the average gene log2(fold change) across the timecourse experiment. Inset highlights the top 35 (10%) dynamic genes. e Heatmap subset from timecourse experiment (panel d) for SNAR-A genes. f – i ChIP-seq track visualization for POLR3G and POLR3GL in THP-1 cells before (left) and after 4h ML-60218 exposure (right) at example tRNA genes tRNA-Asn-GTT (URS0000712B90) ( f ) and tRNA-Tyr-GTA (URS0000755767) ( g ), BCYRN1 ( h ), and SNAR-A genes ( i ). j Moving Venn-diagram overlap analysis of ML-60218 gene sensitivity 4 h post-exposure with diverse experimental results, including dynamic Pol III occupancy (THP-1 +PMA; Fig. ), dynamic RNA abundance (THP-1 +PMA; Fig. ), POLR3G and gene accessibility correlation in primary immune cell differentiation (aggregate cell type analysis; Fig. ), and POLR3G and gene accessibility correlation in primary solid tumors (TCGA; Fig. 4). k Heatmap visualization of maximum enrichment scores, related to Fig. 5j, for individual Pol III-transcribed genes. Inset highlights the subrepertoire of Pol III-transcribed genes that are most likely to be enhanced by subunit POLR3G (maximum enrichment score > = 3). l Model illustration that, compared to POLR3GL, subunit POLR3G enhances the expression of genes encoding specific small noncoding RNA species, including snaR-A, specific tRNAs, and likely BC200 ( BCYRN1 gene). Source Data are provided as a Source Data file.

Article Snippet: THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology).

Techniques: Generated, MTT Cell Proliferation, Control, ChIP-sequencing, Cell Differentiation, Expressing

Journal: Nature Communications

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA

doi: 10.1038/s41467-022-30323-6

Figure Lengend Snippet: a , b Maximum cistromeDB regulatory potential (RP) scores for transcription factors likely to regulate POLR3G ( a ) and POLR3GL ( b ) gene expression. Point size corresponds to number of experiments with top RP score. c Correlation analysis of MYC expression and POLR3G/POLR3GL gene expression ratios in TCGA samples. Spearman’s rank correlation, corresponding p -value computed as two-sided test. P < 2.2e-16. d Volcano plot visualization of mRNA changes following robust knockdown of MYC in RKO cells (Topham et al., 2015) , highlighting POLR3G , POLR3GL , and Pol III subunits mapped in THP-1 cells. e RT-qPCR analysis of MYC , POLR3G , and POLR3GL mRNA, and snaR-A ncRNA levels following MYC KD ( n = 3 biologically independent experiments). P -value = 0.014 ( MYC ), 0.0037 ( POLR3G ), 0.70 ( POLR3GL ), 0.017 (snaR-A seq1), 0.018 (snaR-A seq2). Significance calculated using Student two-sided t -test. f RT-qPCR analysis snaR-A ncRNA levels in THP-1 cells transfected with ASOs targeting conserved sequences within the core (ASO-1) and tail (ASO-2) regions of snaR-A, 48 h post transfection ( n = 4 biologically independent experiments). P -value = 0.00051 (snaR-A seq1), 0.0053 (snaR-A seq2). Significance calculated using Student two-sided t -test. Data are presented as mean ± standard error mean (SEM) from the indicated number of independent samples ( e , f ). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns, nonsignificant. g THP-1 growth curves were generated using MTT cell proliferation assays in THP-1 cells following transfection with control (scramble) or ASO-1 and ASO-2 targeting snaR-A ncRNA. h Model: MYC-driven expression of POLR3G shapes Pol III identity, the availability of subunit POLR3G enhances Pol III activity and transcription potential, downstream expression of snaR-A ncRNA promotes cell viability and proliferation. Differentiation and quiescence are characterized by concomitant loss of MYC, POLR3G, and snaR-A; Pol III transcription potential is thereby restricted in contexts limited to subunit POLR3GL. Source Data are provided as a Source Data file.

Article Snippet: THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control (Catalog# sc-108084, Santa Cruz Biotechnology), [2] control activation (Catalog# sc-437282, Santa Cruz Biotechnology), or [3] POLR3G activation (Catalog# sc-417072-LAC-2, Santa Cruz Biotechnology).

Techniques: Gene Expression, Expressing, Knockdown, Quantitative RT-PCR, Transfection, Generated, MTT Cell Proliferation, Control, Activity Assay