Abstract
Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1,2,3,4,5, are formed by back-splicing of precursor mRNAs in the nucleus6,7,8,9,10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.
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Data availability
Ran HITS–CLIP and circRNA-seq data have been uploaded to the GEO under accession numbers GSE226716 and GSE235899. Uncropped western blots and more detailed figure legends are provided in the Supplementary Information.
Change history
13 March 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41586-024-07281-8
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Acknowledgements
We thank R. Laskey for reading the manuscript; K. Cowley and the staff at the Victorian Centre for Functional Genomics for help with image analysis and the generation of a stable Cas9-expressing CAL51 cell line; the staff at the CCB ACRF Cancer Genomics Facility for assistance with RNA-seq; N. Williamson and the staff at the Bio21 protein characterization facilities for assistance with proteomics and mass photometry; and D. Jans for providing the KPNA2 protein. We acknowledge funding from the NHMRC (1127745 and 2003545 to V.O.W., and 1089167, 1126711 and research fellowship 1118170 to G.J.G.) and the National Breast Cancer Foundation (IN-16-072-2036220). K.A.P. was supported by a Royal Adelaide Hospital Research Committee Florey Fellowship. V.O.W. has been supported by an innovation fellowship from veski and a mid-career fellowship from the Victorian Cancer Agency. V.O.W. thanks R. Wickramasinghe posthumously.
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Contributions
L.H.N. performed the majority of experiments with help from T.W. and K.T.C. A.G.B. prepared samples, analysed sequencing data and performed CLIP-seq. B.K.D. performed CLIP-seq. J.T. analysed circRNA-seq data K.A.P. analysed CLIP-seq data. D.L. prepared spike RNA and samples for sequencing. W.B.H. performed mutagenesis experiments. W.L. purified Ran protein. J.D. and J.A.C. purified IGF2BP1 protein. V.M. purified exportin-2 protein. V.M. and V.O.W. performed and analysed mass-photometry experiments. A.J.D. designed and supervised protein biochemistry and mass photometry. G.J.G. conceived and supervised the study, analysed data and wrote the paper. V.O.W. conceived and supervised the study, performed and analysed experiments and wrote the paper.
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Extended data figures and tables
Extended Data Fig. 1 mRNA export pathways do not function in bulk circRNA export.
a, Cumulative frequency graph of cytoplasmic proportions for the 300 most abundant circRNAs, showing 81% are predominantly localized in the cytoplasm ( > 80% cytoplasmic). A fixed amount of spike circRNA was added to RNA from known proportions of each cytoplasmic and nuclear fraction from cell samples. circRNAs in each sample were quantitated relative to the spike by RNA sequencing. The cytoplasmic and nuclear proportion of each circRNA was then calculated for each cell sample and averaged across 6 cell samples. b,c, Fractionation efficiency was monitored by assessing the levels of MALAT1 and RPS14 RNA (b) and protein levels of Histone H3 and GAPDH (c). Plots are relative to cytoplasmic RNA levels, and show the mean of triplicate readings from 3 independent experiments, ± s.e.m. Protein levels of Histone H3 and GAPDH were monitored in each cellular fraction by western blotting with the indicated antibodies and are representative of 3 independent experiments. d-f, Depletion of mRNA export factors results in a nuclear accumulation and cytoplasmic reduction of a subset of mRNA. Linear mRNA levels were quantitated by qRT-PCR in total cellular (d), nuclear (e) and cytoplasmic (f) RNA extracted from CAL51 cells treated with control, NXF1, ALY or GANP siRNA. g, Depletion of NXF1, GANP, UAP56 and ALY result in nuclear accumulation of poly(A) + RNA. Poly(A) + RNA localization was assessed using a Cy3-labelled oligo dT probe in CAL51 cells treated with control or NXF1, GANP, ALY, UAP56 or URH49 siRNA for 48 (NXF1) or 72 h (GANP, ALY, UAP56, URH49). Nuclei are indicated by DAPI staining (Scale bar, 5 μm) and images are representative of 3 independent depletion experiments. h, Total cellular circRNA levels are broadly unaffected by mRNA export factor depletion. Total cellular circRNA levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated with control or NXF1, GANP, ALY, UAP56 or URH49 siRNA. i,j, UAP56/URH49 depletion has a minimal effect on circRNA export. i, circRNA levels except for circATXN1 and circANKRD17 are not increased in the nucleus following UAP56 depletion. circRNA levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated with control or UAP56 or URH49 siRNA for 72 h. j, UAP56 depletion increases URH49 mRNA levels. UAP56 and URH49 mRNA levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated with control, UAP56 or URH49 siRNA for 72 h. k, Efficiency of UAP56 cross-linking and immunoprecipitation (CLIP) from nuclear extract. UAP56 was immunoprecipitated from nuclear extract of CAL51 cells subjected to UV crosslinking and analysed by western blotting and is representative of 3 independent experiments. l, UAP56 does not interact with circRNA in the nucleus. RNA immunoprecipitated with either a UAP56 antibody or IgG was analysed by qRT-PCR using the indicated primers for circular RNA. For all graphs, statistically significant pair-wise comparisons are indicated (unpaired t-test; * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). All plots are presented as mean of triplicate readings from 3 independent experiments, ± s.e.m.
Extended Data Fig. 2 CRM1 inhibition selectively increases circRNA export.
a, b, CRM1 depletion inhibits ribosomal RNA export. 5 S, 28 S and 18 S rRNA levels were quantitated by either qRT-PCR (a) or gel electrophoresis (b) in RNA extracted from CAL51 cells treated with control or CRM1 siRNA for 72 h. For b, RNA from equivalent numbers of control siRNA and CRM1 siRNA treated cells were analysed. c, CRM1 depletion blocks the nuclear export of Cyclin B1. Immunofluorescence of CAL51 cells treated with control or CRM1 siRNA for 72 h using anti-Cyclin B1 and CRM1 antibodies is shown. Nuclei are indicated by DAPI staining (Scale bar, 5 μm). Nuclear/cytoplasmic intensity of Cyclin B1 was also quantified in individual cells using Cell Profiler (n = 198 (Control siRNA), n = 180 (CRM1 depletion)). d, CRM1 depletion has no effect on poly(A) + RNA localization. Poly(A) + RNA localization was assessed using a Cy3-labelled oligo dT probe in CAL51 cells treated with control or CRM1 siRNA for 72 h. Nuclei are indicated by DAPI staining (Scale bar, 5 μm). e,f, Total cellular circRNA (e) and linear mRNA (f) levels are broadly unaffected by CRM1 depletion. g-j, CRM1 inhibition increases nuclear export of circRNA and reduces nuclear levels of linear DOCK1 mRNA. circRNA (g-h) or linear mRNA (i-j) levels were quantitated by qRT-PCR in nuclear and cytoplasmic RNA extracted from CAL51 cells treated with Selinexor for 12 h. k,l. Total cellular circRNA and linear mRNA levels are broadly unaffected by CRM1 inhibition. Total cellular circRNA (k) or linear mRNA (l) levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated with Selinexor for 12 h. m, CRM1 inhibition blocks the nuclear export of Cyclin B1. Immunofluorescence of CAL51 cells treated with DMSO or Selinexor for one hour using anti-Cyclin B1 and CRM1 antibodies is shown. Nuclei are indicated by DAPI staining (Scale bar, 5 μm). Nuclear/cytoplasmic intensity of Cyclin B1 was also quantified in individual cells using Cell Profiler (n = 218 (DMSO), n = 203 (Selinexor)). n, CRM1 inhibition has no effect on poly(A) + RNA localization. Poly(A) + RNA localization was assessed using a Cy3-labelled oligo dT probe in CAL51 cells treated with DMSO or Selinexor. Nuclei are indicated by DAPI staining (Scale bar, 5 μm). All immunofluorescence images are representative of 3 independent experiments. All plots are presented as mean of triplicate readings from 3 independent experiments, ± s.e.m. For all graphs, statistically significant pair-wise comparisons are indicated (Mann-Whitney test (c,m), unpaired t-test (a,g-l); * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Extended Data Fig. 3 miRNA export pathways do not function in bulk circRNA export.
a, Depletion efficiency of XPO5 was monitored by western blotting with the indicated antibodies. Data are representative of 3 independent experiments. b-d, Nuclear (b), cytoplasmic (c) and total (d) cellular circRNA levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated with control or XPO5 siRNA for 72 h. All plots are presented as mean of triplicate readings from 3 independent experiments, ± s.e.m. For all graphs, statistically significant pair-wise comparisons are indicated (unpaired t-test; * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Identification of factors required for nuclear export of circRNA.
a, Total Ran cellular levels are unchanged by CRM1 depletion. Levels of Ran, CRM1 and actin following CRM1 depletion were monitored by western blotting with the indicated antibodies. b, Sorbitol treatment does not result in a nuclear accumulation of poly(A) + RNA. Poly(A) + RNA localization was assessed using a Cy3-labelled oligo-dT probe in CAL51 cells treated with sorbitol. Nuclei are indicated by DAPI staining (Scale bar, 5 μm). c, Cell-cycle distribution is unaffected by sorbitol treatment. FACS analysis of propidium iodide stained CAL51 cells treated with sorbitol for one hour. The percentage of cells in G1, S, or G2 phase is plotted, showing the mean reading from 3 independent experiments, ± s.e.m. d, Nuclear size is unaffected by sorbitol treatment. Nuclear size was quantitated by cell profiler using DAPI. Median reading with 95% CI is shown for individual cells taken from three independent experiments (n = 328 Control, n = 68, Sorbitol). e, Identification of nuclear proteins that interact with linear or circular SMARCA5 RNA. RNA pulldowns were performed from nuclear extract using either linear biotinylated SMARCA5 RNA or circularized biotinylated SMARCA5 RNA and bound proteins identified by mass-spectrometry. f,g. Generation of SMARCA5 circRNA. f, qRT-PCR analysis with divergent primers that span the splice junction confirmed the presence of circular SMARCA5 RNA. Plots are relative to RNA levels in CAL51 cells (standard), and show the mean of triplicate readings from 3 independent experiments, ± s.e.m. g, Biotinylated circular SMARCA5 RNA was resistant to treatment with RNase R, an exonuclease that degrades linear, but not circular, RNA, whereas linear SMARCA5 was readily digested. Samples were run on an Agilent Tapestation and circular and linear products are indicated. h, Exportin-2 depletion does not result in a nuclear accumulation of poly(A) + RNA. Poly(A) + RNA localization was assessed using a Cy3-labelled oligo dT probe in CAL51 cells treated with control siRNA or Exportin-2 siRNA for 48 h. Nuclei are indicated by DAPI staining (Scale bar, 5 μm). All data are representative of 3 independent experiments. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Depletion of Exportin-2 by two independent methods and in an independent cell line results in a selective nuclear accumulation of circRNA.
a-e. Exportin-2 depletion in HeLa cells results in a selective nuclear accumulation of circRNA. a, Depletion efficiency of Exportin-2 was monitored by western blotting with the indicated antibodies and qRT-PCR using Exportin-2 specific primers. circRNA (b) or linear mRNA (c) levels were quantitated by qRT-PCR in nuclear and cytoplasmic RNA extracted from HeLa cells treated with control or an Exportin-2 siRNA for 72 h. d,e, Total cellular circRNA and linear mRNA levels are broadly unaffected by Exportin-2 depletion. Total cellular circRNA (d) and linear mRNA (e) levels were quantitated by qRT-PCR in RNA extracted from HeLa cells treated with control or Exportin-2 siRNA. f-j. Exportin-2 depletion using an independent siRNA results in a selective nuclear accumulation of circRNA. f, Depletion efficiency of Exportin-2 was monitored by western blotting with the indicated antibodies and qRT-PCR using Exportin-2 specific primers. circRNA (g) or linear mRNA (h) levels were quantitated by qRT-PCR in nuclear and cytoplasmic RNA extracted from CAL51 cells treated with control or an independent Exportin-2 siRNA for 48 h. i,j, Total cellular circRNA and linear mRNA levels are broadly unaffected by Exportin-2 depletion using an independent siRNA. Total cellular circRNA (i) and linear mRNA (j) levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated with control or Exportin-2 siRNA. k-o. Exportin-2 depletion results in a selective nuclear accumulation of circRNA. k, Depletion efficiency of Exportin-2 was monitored by western blotting with the indicated antibodies and qRT-PCR using Exportin-2 specific primers. circRNA (l) or linear mRNA (m) levels were quantitated by qRT-PCR in nuclear and cytoplasmic RNA extracted from stable Cas9-expressing CAL51 cells treated with control (AAVS1 safe harbour region) or pooled Exportin-2 gRNA for 60 h. n,o, Total cellular circRNA and linear mRNA levels are broadly unaffected by Exportin-2 depletion. Total cellular circRNA (n) and linear mRNA (o) levels were quantitated by qRT-PCR in RNA extracted from stable Cas9-expressing CAL51 cells treated with control (AAVS1 safe harbour region) or pooled Exportin-2 gRNA for 60 h. All plots are presented as mean of triplicate readings from 3 independent experiments, ± s.e.m. Plots in (b-e) are from 4 independent experiments, ± s.e.m. For all graphs, statistically significant pair-wise comparisons are indicated (unpaired t-test; * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). All data are representative of 3 independent experiments. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 Effect of depletion of Exportin-2 on circRNA export determined by circRNA sequencing and single molecule FISH.
a, Cell-cycle distribution is unaffected by Exportin-2 depletion. FACS analysis of propidium iodide stained CAL51 cells treated with control siRNA or Exportin-2 siRNA for 48 h. The percentage of cells in G1, S, or G2 phase is plotted, showing the mean reading from 3 independent experiments, ± s.e.m. b, Exportin-2 depletion results in an increase in the nuclear levels of the majority of circRNAs as determined by circRNA sequencing using spike normalization (detailed in methods). Fold change of nuclear and cytoplasmic abundance of 70 of the most abundant circRNAs following Exportin-2 depletion is shown. Fold changes are relative to circRNA read levels in control siRNA-treated cells, assigned an arbitrary value of 1, and show the mean fold change of individual circRNAs in the nucleus or cytoplasm from sequencing of 4 independent siRNA experiments. Median and quartiles are indicated. c,d, circHIPK3 depletion results in a reduction of HIPK3 circRNA levels and an increase in linear HIPK3 mRNA levels as determined by qPCR (c) and single molecule transcript FISH (d). circHIPK3 was depleted using an siRNA targeting the back-splicing junction in CAL51 cells for 48 h. Plots represent the median number of RNA dots/cell per field in control siRNA and circHIPK3 siRNA treated cells quantified using Cell Profiler from three independent depletion experiments. Median and quartiles are indicated. e-h, Exportin-2 depletion results in a selective nuclear accumulation of GDI2 circRNA (hsa_circ_0002665) as determined by qPCR (e) and single molecule transcript FISH (f). Localization of individual circGDI2 RNA and linear GDI2 mRNA transcripts was examined following Exportin-2 depletion (f). Plots in (e) are from 4 independent experiments. Representative images from 3 independent experiments are shown. Nuclear and cytoplasmic RNA dots per cell were quantified using Cell Profiler and are shown in (g and h). At least 573 cells were quantified per condition across three independent experiments. i, Interaction of Exportin-2 with circRNA is regulated by Ran-GTP. Biotinylated RNA pulldown experiments were performed with biotinylated circular SMARCA5 RNA using nuclear extracts from CAL51 cells treated with purified Ran-GTP, and immunoblotted for Exportin-2. Data are representative of 3 independent experiments. j,k, Exportin-2 does not directly interact with linear mRNA or circRNAs in the nucleus. RNA immunoprecipitated with either an Exportin-2 antibody or IgG was analysed by qRT-PCR using the indicated primers for circular RNA (j) and linear mRNA (k). Plots show the mean of triplicate readings from 3 independent CLIP experiments, ± s.e.m. Statistically significant pair-wise comparisons are indicated (Mann-Whitney (d,g,h) or unpaired t-test (c,e); * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 hnRNP interactions with linear and circular SMARCA5 RNA and linear SMARCA5 RNA interactors.
a, hnRNP proteins interact with both linear and circular SMARCA5 RNA. Plots represent hnRNP peptide abundance from 3 independent RNA immunoprecipitation and mass-spectrometry experiments with linear, circular or circular SMARCA5 + Ran-GTP ± s.e.m. b, ALY interacts more strongly with linear RNA. Plots represent ALY peptide abundance from 3 independent RNA immunoprecipitation and mass-spectrometry experiments with linear, circular or circular SMARCA5 + Ran-GTP ± s.e.m. c, NXF1 interacts specifically with linear RNA. d-e. Linear SMARCA5 RNA interactors. Plots represent peptide abundance of the indicated RNA binding proteins (d) and other proteins (e) from 3 independent RNA immunoprecipitation and mass-spectrometry experiments with linear, circular or circular SMARCA5 + Ran-GTP ± s.e.m. Statistically significant pair-wise comparisons are indicated (one way ANOVA followed by Dunnett’s multiple comparisons test; * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Extended Data Fig. 8 IGF2BP1 interacts with circRNA through consensus binding motifs, its depletion reduces levels of some circRNAs and IGF2BP2 interacts with circRNA.
a, Mutation of putative IGF2BP1 binding sites in circularized biotinylated SMARCA5 RNA reduces the retrieval of IGF2BP1 from nuclear extract. Biotinylated RNA pulldown experiments were performed with biotinylated circular SMARCA5 RNA, or mutated circular SMARCA5 RNA (Mut 1 – three binding sites mutated, All mut – 6 binding sites mutated) using nuclear extracts from CAL51 cells, and immunoblotted for IGF2BP1, ALY, and Actin. Nuclear input represents 3% of total IP reaction. b, Depletion efficiency of IGF2BP1 was monitored by western blotting with the indicated antibodies. c, IGF2BP1 depletion reduces total cellular levels of a subset of circRNAs. Total cellular circRNA levels were quantitated by qRT-PCR in RNA extracted from CAL51 cells treated control or IGF2BP1 siRNA for 72 h. d,e, IGF2BP1 depletion reduces nuclear and cytoplasmic levels of a subset of newly synthesized circRNA. Nascent circRNA levels were quantitated by qRT-PCR in nuclear (d) and cytoplasmic (e) RNA as in (c). Plots (c-e) are presented as mean of triplicate readings from 3 (c) or 4 (d,e) independent experiments, ± s.e.m. f, IGF2BP1 depletion results in many circRNAs having decreased levels in both the nucleus and cytoplasm as determined by circRNA sequencing using spike normalization. Fold change of nuclear and cytoplasmic abundance of circRNAs following IGF2BP1 depletion are shown. Fold changes are relative to circRNA read levels in control siRNA-treated cells, assigned an arbitrary value of 1, and show the mean fold change of individual circRNAs in the nucleus or cytoplasm from sequencing of 3 independent siRNA experiments. g, Efficiency of IGF2BP1 cross-linking and immunoprecipitation (CLIP) from nuclear extract. IGF2BP1 was immunoprecipitated from nuclear extract of CAL51 cells and subjected to UV crosslinking and analysed by western blotting. Nuclear input represents 3% of total IP reaction, pulldowns represent 20% of total IP reaction. h, IGF2BP1 interacts with linear mRNA. Immunoprecipitated RNA from (g) was analysed by qRT-PCR using indicated primers for linear mRNA. Plots show mean of triplicate readings from 4 independent CLIP experiments, ± s.e.m. i, Efficiency of IGF2BP2 cross-linking and immunoprecipitation (CLIP) from nuclear extract. IGF2BP2 was immunoprecipitated from nuclear extract of CAL51 cells and subjected to UV crosslinking and analysed by western blotting. Nuclear input represents 3% of total IP reaction, pulldowns represent 20% of total IP reaction. j-k. IGF2BP2 interacts with circRNA. RNA immunoprecipitated with an IGF2BP2 antibody from nuclear extract of CAL51 cells and subjected to UV crosslinking was analysed by qRT-PCR using the indicated primers for circular RNA (j) and linear mRNA (k). Plots show the mean of triplicate readings from 4 independent CLIP experiments, ± s.e.m. All data are representative of 3 independent experiments. For all graphs, statistically significant pair-wise comparisons are indicated (unpaired t-test; * refers to p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 9 Exportin-2 and IGF2BP1 function in circRNA export.
a, Coomassie staining of recombinant proteins used in in vitro binding experiments. b,c, Exportin-2 interacts directly with IGF2BP1 through its RNA binding domains. Recombinant full length and various domains of IGF2BP1 (KH12, KH34 and RRM12) were expressed and purified in E.coli. In vitro binding assays were performed with full length Myc-Exportin-2 incubated with MBP-IGF2BP1-His (full length), MBP-IGF2BP1-His (KH12), MBP-IGF2BP1-His (KH34) and MBP-IGF2BP1-His (RRM12), pre-bound to Ni-NTA agarose. Resin was washed extensively and analysed by SDS polyacrylamide gel electrophoresis, amido black staining and western blotting with Exportin-2 antibody. c, In vitro binding assays were also performed with full length Myc-Exportin-2 (2.5 μg) incubated with MBP-IGF2BP1-His (full length), MBP-IGF2BP1-His (KH12), MBP-IGF2BP1-His (KH34) and MBP-IGF2BP1-His (RRM12) (all 2.5 μg), pre-bound to Ni-NTA agarose. Resin was washed extensively and analysed by SDS polyacrylamide gel electrophoresis and Coomassie staining. d, Exportin-2 can interact with IGF2BP1 in the presence of importin alpha/KPNA2. Similar in vitro binding assays were performed with full length Myc-Exportin-2 pre-incubated for 30 min with GST-KPNA2. This complex was then incubated with MBP-IGF2BP1-His pre-bound to Ni-NTA agarose. Resin was washed extensively and analysed by SDS polyacrylamide gel electrophoresis and Coomassie staining. e, Interaction of IGF2BP1 and Exportin-2 with circRNA is enhanced by Ran-GTP, while its interaction with linear RNA is inhibited by Ran-GTP. Biotinylated RNA pulldown experiments were performed with either biotinylated linear or circular SMARCA5 RNA using nuclear extracts with or without Ran-GTP from CAL51 cells, and immunoblotted for IGF2BP1, Exportin-2 and Actin. Nuclear input represents 3% of total IP reaction. f, Interaction of IGF2BP1 protein with linear RNA is inhibited by Ran-GTP. The effect of addition of Ran-GTP on the interaction of IGF2BP1 and Exportin-2 protein with biotinylated linear SMARCA5 RNA was analysed by immunoblotting for Exportin-2, IGF2BP1 and Ran. g, IGF2BP1 interacts with circRNA in the absence of Exportin-2. Using constant amounts of biotinylated SMARCA5 circRNA, the interaction of IGF2BP1, Exportin-2 and Ran-GTP proteins with circRNA was analysed by immunoblotting for Exportin-2, IGF2BP1 and Ran. All data are representative of 3 independent experiments. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 10 Characterization of Ran, IGF2BP1 and Exportin-2 function in circRNA export.
a. Coomassie staining of recombinant proteins used in mass photometry experiments. b-c, Purified Exportin-2 (b) and IGF2BP1 (c) run as monomers as measured by mass photometry. Calculated molecular masses of individual proteins are shown. d-h, Ran HITS-CLIP identifies selective binding of circRNAs. Phosphorimages of RNA-protein complexes captured by Ran immunoprecipitation and run on SDS-PAGE after digestion with high or low concentration of RNase 1 and 32P-labelling (d,e). The regions cut from the gel for HITS-CLIP are labelled. f, Phosphorimage of urea gel of RNA from each region of the low RNase SDS-PAGE gel in (e), showing the region eluted for RNA sequencing. The linkers add 57nt to the RNA length. g, Efficiency of Ran cross-linking and immunoprecipitation (CLIP). Ran was immunoprecipitated from nuclear extract of CAL51 cells subjected to UV crosslinking and analysed by immunoblotting for Ran (green) and tubulin (red). h, Reads per kilobase per million reads mapped (RPKM) for the host genes of the most abundant 250 circRNAs. Read coverage was calculated in circle-producing exons (‘circRNA’), non-circRNA-producing exons (‘noncirc’), 3’-UTRs and introns. The line shown is the median. The QKI HITS-CLIP data are from72 and Ago HITS-CLIP data from73. i, Ran and IGF2BP1 interact with each other in vivo in the nucleus. Endogenous Ran was immunoprecipitated from nuclear extract of CAL51 cells and immunoblotted for IGF2BP1 and Ran. Nuclear input represents 3% of total IP reaction. j, Ran interacts directly with IGF2BP1. In vitro binding assays were performed with full length Myc-IGF2BP1 (1 μg) incubated with Ran-His (full length, 5 μg), pre-bound to Ni-NTA agarose. Resin was washed extensively and analysed by SDS polyacrylamide gel electrophoresis, amido black staining and western blotting with IGF2BP1 antibody. k, Model describing a nuclear export pathway involved in the transport of circular RNA that is dependent on Ran-GTP. This pathway requires Exportin-2 as an export receptor, IGF2BP1 as an adaptor protein that physically interacts with circRNA and Exportin-2, and Ran-GTP which interacts with circRNA, Exportin-2 and IGF2BP1. In the cytoplasm, the circRNA export cargo is released upon conversion of Ran-GTP into Ran-GDP. All data are representative of 3 independent experiments. For gel source data, see Supplementary Fig. 1.
Supplementary information
Supplementary Information
Detailed figure legends for Figs. 1–5 and uncropped western blots.
Supplementary Table 1
circRNA and linear mRNA abundance.
Supplementary Table 2
Nuclear circRNA and linear RNA interactor peptide abundance.
Supplementary Table 3
Top 70 abundant circRNAs analysed from sequencing of nuclear and cytoplasmic fractions.
Supplementary Table 4
siRNA sequences.
Supplementary Table 5
Oligos and primers.
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Ngo, L.H., Bert, A.G., Dredge, B.K. et al. Nuclear export of circular RNA. Nature 627, 212–220 (2024). https://doi.org/10.1038/s41586-024-07060-5
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DOI: https://doi.org/10.1038/s41586-024-07060-5
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