Abstract
Circular RNA (circRNA) is covalently closed, single-stranded RNA produced by back-splicing. A few circRNAs have been implicated as functional; however, we lack understanding of pathways that are regulated by circRNAs. Here we generated a pooled short-hairpin RNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at different levels (ranging from low to high) in humans. We used this library for loss-of-function proliferation screens in a panel of 18 cancer cell lines from four tissue types harbouring mutations leading to constitutive activity of defined pathways. Both context-specific and non-specific circRNAs were identified. Some circRNAs were found to directly regulate their precursor, whereas some have a function unrelated to their precursor. We validated these observations with a secondary screen and uncovered a role for circRERE(4–10) and circHUWE1(22,23), two cell-essential circRNAs, circSMAD2(2–6), a WNT pathway regulator, and circMTO1(2,RI,3), a regulator of MAPK signalling. Our work sheds light on pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function.
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Data availability
All raw and processed data from all screens are available in the Supplementary Tables. All plasmids used in this paper are available through Addgene. Source data are provided with this paper.
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Acknowledgements
This work was supported by an Australian Research Council grant (grant number DP210101755 to J.R., G.G. and P.T.). J.R. is supported by a Victoria Cancer Agency fellowship (grant number MCRF20035). S.J.C. is supported by an Investigator Grant (Leadership; grant number GNT1198014) awarded by the National Health and Medical Research Council. M.N. is supported by a Tour de-Cure PhD top-up scholarship (grant number RSP-145-FY2023). We thank the Functional Genomics Platform, the Bioinformatics Platform and Micromon genomics platform at Monash University for help with loss-of-function screens and data analysis. We thank T. Jarde and H. Abud from the Monash Organoid Platform for their help with the organoid experiments. We thank M. Lazarou from the Walter and Eliza Hall Institute for helpful discussions and suggestions.
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Conceptualization: J.R., G.J.G. and S.J.C. Methodology: J.R., L.L., M.N., X.X., J.P., A.H., S.J.C. and J.Z. Analysis: J.R., P.T., L.P.-J., L.L. and M.N. Writing—original draft: J.R., M.N., G.J.G., S.J.C. and L.L. Writing—review and editing: all authors. Supervision: J.R., L.L. and J.Z. Funding acquisition: J.R., G.J.G. and P.T. The authors read and approved the final manuscript.
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Extended data
Extended Data Fig. 1 Primary loss of function screen identifies context-specific and non-specific circRNAs.
(a) Overlap between circRNAs profiled in this study and circRNAs that were profiled in previous prostate specific circRNA screens10. (b) Distribution of proliferation changes following shRNA mediated knockdown of core-essential genes. (c) Comparison of proliferation changes (average fold change across all cell lines) in the 939 overlapping circRNAs from this study and from Chen et al.10 pValue calculated using Pearson correlation. (d) Pearson correlation in proliferation changes observed following suppression of the circRNA or the linear mRNA using shRNAs or CRISPRs. Gene dependency scores were taken from DepMap61. (e) Volcano plots showing two class comparisons that identify pathway and lineage specific circRNAs. pValue calculated using a one tail T.test with unequal variance. Source numerical data (C,D,E) are available in source data.
Extended Data Fig. 2 Secondary screen validates context-specific and non-specific circRNAs.
(a) Sequencing reads (each read represents one cell) in all replicates from primary and secondary screens. Each dot represents a different replicate. Centre line represents the mean. (b) Distribution of proliferation changes following shRNA mediated knockdown of core-essential genes.
Extended Data Fig. 3 Identification circHUWE1(22,23) as a core-essential circRNAs.
(a) Crystal violet images of cells 7-DPI with shRNAs targeting linear or circular HUWE1. (b) Crystal violet images of normal immortalized cells 7-DPI with shRNAs targeting linear or circular HUWE1. (c) Expression of circHUWE1(22,23) or linearized circHUWE1(22,23) (not containing the back-splice intron) in HEK293T cells transfected with an overexpression vector expressing; GFP or circHUWE1(22,23) or linearized circHUWE1(22,23) (without the back-splice intron). Linear circHUWE1(22,23) primers detect the levels of both linear and circHUWE1(22,23). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 3.20e-02 (circHUWE1(19,20) OE, circular), 0.28 (linearHUWE1(19,20) OE, circular), 7.04e-02 (circHUWE1(19,20) OE, linear), 7.90e-03 (linearHUWE1(19,20) OE, linear). (d) Crystal violet images of proliferation of RKO cells overexpressing GFP or circHUWE1(22,23) or linearized circHUWE1(22,23) (without back-splice intron) 7-DPI with control or a circHUWE1(22,23) targeting shRNA. (e) Proliferation changes from DepMap61 following CRISPR mediated suppression of HUWE1 gene. Box limits indicate upper and lower quartiles, and whiskers represent the minimum and maximum values, centre lines show the median. pValue was calculated using a one-tailed unpaired T-test. (f) Proliferation changes from DepMap27 following shRNA mediated suppression of HUWE1 gene. Box limits indicate upper and lower quartiles, and whiskers represent the minimum and maximum values, centre lines show the median. pValue was calculated using a one-tailed unpaired T-test. NS, not significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Source numerical data (C) are available in source data.
Extended Data Fig. 4 Identification circRERE(4–10) as a core-essential circRNA.
(a) Crystal violet images 7-DPI with shRNAs targeting circular or linear RERE in a panel of cancer cell lines. (b) Crystal violet images 7-DPI of shRNAs targeting circular or linear RERE in two normal immortalized cell lines. (c) Proliferation changes from DepMap61 of individual shRNAs following suppression of linear RERE gene. Box limits indicate upper and lower quartiles, and whiskers represent the minimum and maximum values, centre lines show the median. pValue was calculated using a one-tailed unpaired T-test. (d) Expression of circRERE(4-10) or linearized circRERE(4-10) (not containing the back-splice intron) in HEK293T cells transfected with an overexpression vector expressing; GFP or circRERE(4-10) or linearized circRERE(4-10) (without the back-splice intron). Linear circRERE(4-10) primers detect the levels of both linear and circRERE(4-10). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/− SD, n=3 biological replicates. (P = 5.67e-02 (circRERE(4,11), circular), 0.2 (linear circRERE(4,11), circular), 5.73e-02 (circRERE(4,11), linear), 7.98e-03 (linear circRERE(4,11), linear)). (e) Crystal violet images of proliferation in RKO cells overexpressing GFP or circRERE(4-10) or linearized circRERE(4-10) (without back-splice intron) 7-DPI with control or a circRERE(4-10) targeting shRNA. (f) Quantification of live dead cells using trypan blue staining in RKO cells expressing shRNA targeting circRERE(4-10) treated with the indicated cell death inhibitors. (g) Quantification of live dead cells using trypan blue staining in RKO cells expressing shRNA targeting linear or circular RERE treated with DMSO or the Ferroptosis inhibitor Ferrostatin-1. (h) Lipid oxidation in HA1EM cells treated with a Ferroptosis activator (RSL3) with or without a Ferroptosis inhibitor (Ferrostatin-1). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.32 (DMSO), 8.24e-05 (0.1µM), 6.21e-04 (0.5µM), 4.08e-07 (1µM)). (i) Lipid oxidation measured in HA1EM cells treated with a Ferroptosis activator (RSL3) with or without overexpression of circRERE(4-10). pValue was calculated using a one-tailed unpaired T-test. (P = 0.3 (DMSO), 6.99e-05 (0.1µM), 7.80e-04 (0.5µM), 1.65e-02 1µM)). Data are mean +/−SD, n=3 biological replicates. NS, not significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Source numerical data (D,F-I) are available in source data.
Extended Data Fig. 5 Identification of circRNAs that regulate the WNT signalling pathway.
(a) Expression of linear or circular ZNF652 4 DPI with circZNF652(4,RI,5) targeting shRNAs in DLD1 cells. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=2 biological replicates. (P = 1.62e-03 (linear), 2.36e-03 (circular)). (b) Expression of linear or circular PKHD1 4-DPI with circPKHD1(28,29,34,35) targeting shRNAs in DLD1 cells. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=2 biological replicates. (P = 0.35 (linear), 1.68e-03 (circular)). (c) Crystal violet images 7 DPI with shRNAs targeting circSMAD2(2–6) in WNT active or inactive cell lines. (d) Crystal violet proliferation assays 7 DPI with shRNAs targeting circular or linear ZNF652. (e) Quantification of proliferation changes induced by shRNAs targeting circZNF652(4,RI,5). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.26, 0.04 (RKO), 0.04, 0.02 (LN229), 1.3e-06, 2.1e-06 (DLD1), 0.003, 0.002 (HT29), 2.51e-06, 1.38e-05(SW620)). (f) Quantification of proliferation changes induced by shRNAs targeting linear ZNF652 mRNA 7-DPI. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.11, 0.27 (RKO), 0.37, 0.08 (LN229), 0.07, 0.03 (DLD1), 0.23, 0.29 (HT29), 0.03, 0.13 (SW620)). (g) Crystal violet images 7-DPI with shRNAs targeting circular or linear PKHD1 in WNT active or inactive cell lines. (h) Quantification of proliferation changes induced by shRNAs targeting circPKHD1(28,29,34,35). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.31, 0.38 (RKO), 0.16, 0.36 (LN229), 2.42e-07, 1.57e-05 (DLD1), 7.45e-05, 3.41e-05 (HT29), 9.65e-05, 1.88e-04 (SW620)). (i) Quantification of proliferation changes induced by shRNAs targeting linear PKHD1 mRNA. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.23, 0.49 (RKO), 0.20, 0.08 (LN229), 0.32, 0.23 (DLD1), 0.36, 0.09 (HT29), 0.33, 0.10 (SW620)). (j) Expression of circSMAD2(2–6) or linearized circSMAD2(2–6) (not containing the back-splice intron) in HEK293T cells transfected with an overexpression vector expressing; GFP or circSMAD2(2–6) or linearized circSMAD2(2–6) (without the back-splice intron). Linear circSMAD2(2–6) primers detect the levels of both linear and circSMAD2(2–6). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 3.18e-02 (circSMAD2(2–6), circular), 0.33 (linear circSMAD2(2–6), circular), 6.97e-02 (circSMAD2(2–6), linear), 3.55e-02 (linear circSMAD2(2–6), linear)). (k) Schematic of possible circSMAD2(2–6) products produced from the circRNA overexpression vector. (l) Agarose gel showing the size of PCR products from control cells or cells containing the circSMAD2(2–6) overexpression vector. (m) Crystal violet images of proliferation in DLD1 cells overexpressing GFP or circSMAD2(2–6) or linearized circSMAD2(2–6) (without back-splice intron) 7-DPI with control or a circSMAD2(2–6) targeting shRNA. (n) Crystal violet images showing proliferation of DLD1 (WNT active) or RKO (WNT inactive) cells overexpressing CTNNB1 7-DPI with control or circSMAD2(2–6) targeting shRNAs. (o) CTNNB1 protein levels in samples from (N). NS, not significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Source numerical data (A,B,E,F,H,I,J) and unprocessed blots (O) are available in source data.
Extended Data Fig. 6 Validation of circRNAs that are essential for KRAS mutant cell lines.
(a) Expression of linear or circular RNF170 4-DPI with linear or circular RNF170 targeting shRNAs in MIAPACA2 cells. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 4.22e-04, 0.17 (linear), 3.77e-02, 2.68e-04 (circular)). (b) Proliferation of KRAS mutant or WT cells 7-DPI with shRNAs targeting circular or linear RNF170. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.37, 0.22, 0.01, 4.84e-03 (DLD1), 0.44, 0.31, 3.64e-04, 1.94e-03 (MIAPACA2), 0.33, 0.29, 0.38, 0.37 (RKO), 0.27, 0.40, 0.19, 0.03 (LN229)). (c) Expression of linear or circular NOL12(2,4) 4-DPI with linear or circular NOL12 targeting shRNAs in MIAPACA2 cells. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 5.25e-08, 0.13 (linear), 4.47e-02, 9.51e-10 (circular)). (d) Proliferation of KRAS mutant or WT cancer cells 7-DPI with shRNAs targeting circular or linear NOL12. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 4.47e-03, 6.38e-03, 6.74e-03, 7.97e-03 (DLD1), 3.74e-03, 4.42e-03, 9.84e-04, 1.44e-04 (MIAPACA2), 5.62e-04, 6.66e-05, 2.72e-03, 3.90e-03 (RKO), 1.51e-05, 2.14e-03, 0.16, 0.5 (LN229)). (e) Expression of linear or circular NUDC(5,6) 4 DPI with linear or circular NUDC targeting shRNAs in MIAPACA2 cells. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 1.24e-02, 4.73e-02 (linear), 0.17, 1.75e-03 (circular)). (f) Proliferation of KRAS mutant or WT cancer cells 7-DPI with shRNAs targeting circular or linear NUDC. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 3.91e-04, 1.66e-03, 1.41e-03, 3.45e-03 (DLD1), 5.24e-05, 2.75e-04, 1.15e-04, 3.69e-04 (MIAPACA2), 1.31e-03, 4.74e-04, 3.90e-02, 8.50e-02 (RKO), 1.79e-03, 1.68e-02, 1.73e-03, 0.31 (LN229)). (g) Levels of pERK and pAKT following suppression of circRNF170(2–6) in KRAS WT and KRAS mutant cells. (h) Levels of pERK and pAKT following suppression of circNOL12(2–4) in KRAS WT or mutant cells. NS, not significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Source numerical data (A-F) and unprocessed blots (G,H) are available in source data.
Extended Data Fig. 7 Identification of circMTO1(2,RI,3) as a regulator of BRAF dependent cancers.
(a) Crystal violet images following introduction of shRNAs targeting circMTO1(2,RI,3) in BRAF dependent or independent cell lines. (b) Expression of circMTO1(2,RI,3) or linearized circMTO1(2,RI,3) (not containing the back-splice intron) in HEK293T cells transfected with an overexpression vector expressing; GFP or circMTO1(2,RI,3) or linearized circMTO1(2,RI,3) (without the back-splice intron). Linear circMTO1(2,RI,3) primers detect the levels of both linear and circMTO1(2,RI,3). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 0.01, 0.4 (circular), 5.52e-02, 4.99e-03 (linear)). (c) Crystal violet images of proliferation in A375 cells overexpressing GFP or circMTO1(2,RI,3) or linearized circMTO1(2,RI,3) (without back-splice intron) 7-DPI with control or a circMTO1(2,RI,3) targeting shRNA. (d) Proliferation of C32 (BRAF mutated) cells following treatment with a BRAF inhibitor (PLX4032) and circMTO1(2,RI,3) targeting shRNAs. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P = 4.35e-04, 4.14e-04, 3.75e-04 (shLacZ), 9.00e-03, 0.18, 7.94e-04 (shcircMTO1(2,RI,3)_1), 2.41e-03, 3.24e-03, 8.80e-05 (shcircMTO1(2,RI,3)_2)). (e) Proliferation of WM266-4 (BRAF mutated) cells following treatment with a BRAF inhibitor (PLX4032) and circMTO1(2,RI,3) targeting shRNAs. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates). (P = 1.11e-04, 1.19e-04, 3.50e-06 (shLacZ), 0.35, 2.41e-03, 6.97e-02, (shcircMTO1(2,RI,3)_1), 1.32e-03, 5.13e-04, 9.06e-05 (shcircMTO1(2,RI,3)_2)). NS, not significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Source numerical data (B,D,E) are available in source data.
Extended Data Fig. 8 Knockdown of circMTO1(2,RI,3) inhibits pERK or pAKT depending on the levels of PTEN.
(a) Quantification of pAKT protein levels following suppression of circMTO1(2,RI,3). Each dot represents an independent WB. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=2 biological replicates. (P = 0.04, 0.07 (SKMEL28), 0.06, 0.02 (C32), 0.02, 0.10 (WM266-4), 0.05, 0.04 (Malme-3M), 0.05, 0.07 (A375), 0.11, 0.09 (HT29), 0.18, 0.17 (DLD1)). (b) Quantification of pERK protein levels following suppression of circMTO1(2,RI,3). Each dot represents an independent WB. pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=2 biological replicates. (P = 0.45, 0.44 (SKMEL28), 0.35, 0.28 (C32), 0.10, 0.09 (WM266-4), 0.04, 0.07 (Malme-3M), 0.02, 3.33e-3 (A375), 0.02, 0.05 (HT29), 0.07, 0.05 (DLD1)). (c) Partial rescue of proliferation in WM266-4 cells following treatment with an AKT activator (SC79) and shRNAs targeting circMTO1(2,RI,3). pValue was calculated using a one-tailed unpaired T-test. Data are mean +/−SD, n=3 biological replicates. (P =0.08, 0.07 (shLacZ), 6.84e-06, 2.09e-06 (shcircMTO1(2,RI,3)_1), 3.86e-05, 5.67e-07 (shcircMTO1(2,RI,3)_2)). (d) Rescue of circMTO1(2,RI,3) inhibition in pAKT levels following treatment with SC79 in WM266-4 cells. NS, not significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Source numerical data (A-C) and unprocessed blots (D) are available in source data.
Supplementary information
Supplementary Tables 1–12
Supplementary Table 1. Genomic features of circRNAs that used in this study. Genomic coordinates and read counts of circRNAs that were used in this study. Supplementary Table 2. Sequences of shRNAs used in pooled primary screens. Target sequences of shRNAs targeting circRNAs or core essential genes that were used in primary screens. Supplementary Table 3. Read numbers from the primary screen. Read numbers for the shRNA pool or cells infected with circRNA-targeting library 21 d.p.i. Supplementary Table 4. Fold change calculations in the primary circRNA screen. The log2-transformed fold change in circRNA abundance was calculated by comparing shRNA abundance after 21 days in culture to the DNA pool and using the average of all five shRNAs targeting each circRNA. Supplementary Table 5. Correlation between gene dependency from DepMap and circRNA dependency. For each circRNA, the Pearson’s correlation between the proliferation effect of the linear (CRISPR or shRNA from refs. 27,55) and circRNA on proliferation was calculated. Supplementary Table 6. Genes that score in two class comparisons in primary screens. For each two-class comparison, the difference between classes was determined by calculating the difference in viability between two classes. Supplementary Table 7. Sequences of shRNAs used in pooled secondary screens. Target sequences of shRNAs targeting circRNAs or control genes that were used in secondary validation screens. Supplementary Table 8. Read numbers from the secondary screen. Read numbers for the shRNA pool or cells infected with circRNA-targeting library 21 d.p.i. Supplementary Table 9. Fold change calculations for the secondary circRNA screen. The log2-transformed fold change in circRNA abundance was calculated by comparing shRNA abundance after 21 days in culture to the DNA pool and using the average of all five shRNAs targeting each circRNA. Supplementary Table 10. Genes that score in two class comparisons in secondary screens. For each two-class comparison, the difference between classes was determined by calculating the difference in viability between two classes. Supplementary Table 11. Primers and shRNAs used in this study. Supplementary Table 12. STR analysis of the cell lines used in this study.
Source data
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 5
Unprocessed western blots.
Source Data Fig. 6
Unprocessed western blots.
Source Data Fig. 7
Unprocessed western blots.
Source data Figs. 1,3–5 and Extended Data Figs. 3–8
Statistical source data.
Source data Extended Data Figs. 5o, 6g,h and 8d
Unprocessed western blot images from extended data figures.
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Liu, L., Neve, M., Perlaza-Jimenez, L. et al. Systematic loss-of-function screens identify pathway-specific functional circular RNAs. Nat Cell Biol 26, 1359–1372 (2024). https://doi.org/10.1038/s41556-024-01467-y
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DOI: https://doi.org/10.1038/s41556-024-01467-y
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