HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, United States
- Center for Therapeutics Discovery, department of Oncological Sciences and Pharmacological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, United States
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, United States
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, United States
- IIGM (Italian Institute for Genomic Medicine), Italy
- Dipartimento di Scienze della Vita e Biologia dei Sistemi Università di Torino, Italy
- Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Netherlands
- Cancer Science Institute of Singapore, National University of Singapore, Singapore
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- NTU Institute of Structural Biology, Nanyang Technological University, Singapore
- Department of Medical Biophysics, University of Toronto, Canada
- Princess Margaret Cancer Center, University Health Network, Canada
- Ontario Institute for Cancer Research, Canada
- Interdisciplinary Melanoma Cooperative Group, New York University Langone Medical Center, United States
- Department of Pathology, New York University Langone Medical Center, United States
- Department of Medicine, Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, United States
- Department of Pathology, Icahn School of Medicine at Mount Sinai, United States
- Max Delbruck Center for Molecular Medicine, Germany
- Department of Molecular Oncology, Moffitt Cancer Center, United States
Figures
Figure 1 with 1 supplement
A pooled shRNA screen identifies HNRNPM as a regulator of prostate cancer (PCa) cell growth.
(A) Schematic of overall experimental design. (B) Multidimensional scaling (MDS) plot of individual samples (in vitro passaged cells and tumors) collected in the screen, in relation to each other. (C…
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Figure 1—source data 1
Rotational Gene Set Analysis (ROAST) results: tumor versus input (P1).
- https://cdn.elifesciences.org/articles/59654/elife-59654-fig1-data1-v2.xlsx
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Figure 1—source data 2
Rotational Gene Set Analysis (ROAST) results: in vitro.
- https://cdn.elifesciences.org/articles/59654/elife-59654-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Verification of top hits in the pooled shRNA screen.
(A) Heatmap showing overall hairpin enrichment over time in in vitro passaged LNCAP cells (left), in vivo LNCAP xenografts (center), as well as input PLKO.1 plasmid and lentiviral pools (right). Colo…
Figure 2 with 2 supplements
HNRNPM inhibits prostate cancer (PCa) cell growth in vitro and in vivo.
HNRNPM RNA (A) and protein levels (B) upon expression of scrambled (Scr) or HNRNPM-specific shRNAs (2B7, 2B9, 2B10) in LNCAP cells. UT: untreated cells. (C) Cell proliferation assays of LNCAP cells …
Figure 2—figure supplement 1
Impact of HNRNPM deficiency in independent prostate cancer (PCa) cell lines.
(A) Heatmap showing relative enrichment and depletion of HNRNPM-specific shRNAs in the screen. (B) HNRNPM RNA and (C) protein levels upon expression of scrambled or HNRNPM-specific shRNAs (2B7, 2B9, …
Figure 2—figure supplement 2
Xenograft tumor growth over time.
(A) LNCAP cells treated with scrambled (SCR; black lines) or HNRNPM-specific shRNAs (2B7; orange lines or 2B9; red lines) (n = 11 for all three conditions) per injected into the flanks of CB17-SCID …
Figure 3 with 1 supplement
HNRNPM binds guanidine and uridine (GU)-rich elements within long introns.
(A) Normalized read density of HNRNPM eCLIP at the HNRNPM and PRAM1 genes. Input (yellow) and immunoprecipitation (red) tracks are shown. High-confidence (p<0.05, fold enrichment >2) peaks are shown …
Figure 3—figure supplement 1
eCLIP analysis of HNRNPM-binding sites (associated with Figure 3).
(A, left) Immunoprecipitation-western blot validation of anti-HNRNPM antibody pull-down efficiency in UV-crosslinked LNCAP lysates. (A, center) Scan of membrane showing regions excised for eCLIP …
Figure 4
HNRNPM depletion results in increased exon inclusion.
(A) Scatterplot of all significantly changed (p<0.05, FDR < 0.05) genes (gray point) in HNRNPM shRNA-treated cells, and their HNRNPM-binding status. HNRNPM-bound genes are highlighted in orange. Red …
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Figure 4—source data 1
Gene expression changes in all HNRNPM-bound genes.
- https://cdn.elifesciences.org/articles/59654/elife-59654-fig4-data1-v2.xlsx
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Figure 4—source data 2
Binding status of all significantly differentially expressed genes in HNRNPM knockdown.
- https://cdn.elifesciences.org/articles/59654/elife-59654-fig4-data2-v2.xlsx
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Figure 4—source data 3
Binding status of significantly changed skipped exon events.
- https://cdn.elifesciences.org/articles/59654/elife-59654-fig4-data3-v2.xlsx
Figure 5 with 1 supplement
HNRNPM depletion results in increased circular RNA (circRNA) formation.
(A) Scatterplot of circRNA events that are significantly (p<0.05, FDR < 0.05) changed in sh2B7 or sh2B9-treated cells compared to scrambled shRNA-treated cells. All individual captured events are …
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Figure 5—source data 1
Binding status of significantly changed circular RNAs.
- https://cdn.elifesciences.org/articles/59654/elife-59654-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
A subset HNRNPM-regulated circular RNA (circRNA) significantly predicts patient survival (associated with Figure 5).
(A) Biochemical relapse survival curves of prostate cancer patients expressing high (>80th percentile), low (≤20th percentile), or mid (20th–80th percentile) levels of the indicated circRNAs. Shown …
Figure 6 with 3 supplements
Mimicking HNRNPM-dependent linear-splicing events in cells inhibits exon inclusion and cell growth.
(A, top panel) Schematic showing outcomes of splicing reporter assay. Wildtype (WT) or mutant (MUT) HNRNPM-binding sites at the USP33 or APMAP genes and identified by eCLIP were cloned into a …
Figure 6—figure supplement 1
HNRNPM-bound sequences inhibit exon inclusion.
(A) Plots showing HNRNPM-bound regions of the USP33 and APMAP gene that were cloned and inserted into the minigene construct. (B) Representative flow cytometry plots showing differential splicing …
Figure 6—figure supplement 2
Structural characterization of HNRNPM-bound introns.
(A) Distribution of unique HNRNPM peaks found in the immediate flanking upstream and downstream introns of mis-spliced linear (top-left panel) or circular (top-right panel) mis-splicing events that …
Figure 6—figure supplement 3
HNRNPM regulates a multigenic splicing program to maintain cell proliferation.
(A) Distribution of predicted outcomes of HNRNPM-dependent linear-splicing events occurring in coding domains of affected genes. (B) Structure of the HNRNPM-regulated WD40 domain in wildtype EED …
Figure 7 with 1 supplement
HNRNPM regulates growth and splicing in other cancer cell lines.
(A) Proliferation curves of PANC1, Calu3, Mel501, SKMEL147, and HepG2 cells, representing cell lines derived from pancreatic adenocarcinoma, lung adenocarcinoma, melanoma, and hepatoblastoma, upon …
Figure 7—figure supplement 1
HNRNPM mRNA levels are negatively correlated with target circular RNA (circRNA) levels in melanoma short-term cultures.
(A) Short-term cultures were subjected to RNA-sequencing following rRNA removal. BM: brain metastasis sample; LN: lymph node metastasis; Par: parotid gland metastasis. Numbers of short-term cultures …
Author response image 1
Analysis of EED exon10 inclusion in TCGA patient samples.
(a) To stratify patients between “high hnrnpm” and “low hnrnpm” expression, we used a 90th percentile cutoff within tumor samples. This cutoff was chosen based on HnRNPM expression in normal …
Author response image 2
Increased inclusion of single HNRNPM-regulated exons poorly correlates with improved patient survival.
Disease free survival plot of TCGA PRAD patients, stratified by the total number of events per patient where the PSI of the indicated HNRNPM-regulated exon exceeds that of the median PSI within the …
Author response image 3
Combined knockdown of multiple splicing events results in inhibition of cell proliferation.
LNCAP cells were transfected with 2OMe SSOs targeting ZNF548 (ZNF548 SSO-3), PRKAB2 (PRKAB SSO-2) and EED (EED SSO-1). To adjust for overall SSO concentrations in cells, control (SCR) targeting SSOs …
Author response image 4
EED overexpression in HNRNPM knockdown cells.
LNCAP cells were transduced with either a control (empty vector) or EED overexpressing lentivirus (Blasticidin resistant). After selection, cells were transduced with lentivirus expressing either …
Additional files
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Supplementary file 1
Table of shRNAs and sequences used for pooled screens.
- https://cdn.elifesciences.org/articles/59654/elife-59654-supp1-v2.xlsx
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Supplementary file 2
Sequences used for bichromatic splicing reporter.
- https://cdn.elifesciences.org/articles/59654/elife-59654-supp2-v2.xlsx
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Supplementary file 3
Sequences of RNA oligos used for RNA G-quadruplex hemin assays.
- https://cdn.elifesciences.org/articles/59654/elife-59654-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/59654/elife-59654-transrepform-v2.docx
