Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans

Lesca M Holdt, Anika Stahringer, Kristina Sass, Garwin Pichler, Nils A Kulak, Wolfgang Wilfert, Alexander Kohlmaier, Andreas Herbst, Bernd H Northoff, Alexandros Nicolaou, Gabor Gäbel, Frank Beutner, Markus Scholz, Joachim Thiery, Kiran Musunuru, Knut Krohn, Matthias Mann, Daniel Teupser, Lesca M Holdt, Anika Stahringer, Kristina Sass, Garwin Pichler, Nils A Kulak, Wolfgang Wilfert, Alexander Kohlmaier, Andreas Herbst, Bernd H Northoff, Alexandros Nicolaou, Gabor Gäbel, Frank Beutner, Markus Scholz, Joachim Thiery, Kiran Musunuru, Knut Krohn, Matthias Mann, Daniel Teupser

Abstract

Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease.

Figures

Figure 1. CircANRIL expression in human vascular…
Figure 1. CircANRIL expression in human vascular tissue and association with atheroprotection at 9p21.
(a) Schematic of circANRIL at 9p21 and qPCR assays for isoform specific quantification. CircANRIL contains exons 5, 6 and 7. Exon 7 is non-canonically spliced to exon 5. (b) qPCR analysis of circANRIL expression in human tissues, primary cells and cell lines. Beta actin (ACTB), house-keeping gene; FB, adventitial fibroblasts; HEK, HEK-293 cell line; THP1 and MonoMac, human monocytic cell line. Analyses were done in RNA pools of at least three donors. (c) In situ hybridization of circANRIL in human atherosclerotic plaque and co-localization with smooth muscle actin (SMA)-positive cells and macrophages (CD68). Sense control (CTR)-negative control. Representative staining out of three biological replicates. (d) Association of circANRIL with 9p21 haplotypes in PBMC from CAD patients. Protective (A, n=498), heterozygote (H, n=979) and risk (G, n=456) haplotypes. (e) Association of circANRIL with 9p21 haplotypes A (n=49), H (n=114) and G (n=55) in endarterectomy specimens. (f) Association of circ/linANRIL ratio in PBMC with severity of CAD (No, n=745; <50% stenosis, n=392; ≥50% stenosis, n=747). Data are given as mean±s.e.m., and associations were calculated using robust linear regression models.
Figure 2. Atherosclerosis-related cell functions in c…
Figure 2. Atherosclerosis-related cell functions in circANRIL-overexpressing cells.
(a) Schematic of the vector construct for circANRIL stable and transient overexpression. RNA polymerase II (RNAP II). (b) ANRIL isoform expression in HEK-293 cells that stably express circANRIL, linANRIL or empty vector (three biological replicates each). PCR of reverse-transcribed RNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), house-keeping gene; OdT, oligo(d)T primers; RH, random hexamer primers. (c) Apoptosis and (d) proliferation in HEK-293 cell line stably overexpressing circANRIL (pool of 3 biological replicates, and 8 and 12 technical replicates per condition, respectively), compared with overexpression of an unrelated circular RNA (circHPRT1). Apoptosis (e) and proliferation (f) after RNAi against circANRIL or non-circANRIL or scrambled (SCR) siRNA control. Quadruplicate measurements per condition. *P<0.05; **P<0.01; ***P<0.001. Comparison of multiple groups was done using analysis of variance, and Tukey was performed as post hoc test in c,e. Two-tailed unpaired Student's t-test in d,f. Data are given as mean±s.e.m.
Figure 3. Role of endogenous circANRIL on…
Figure 3. Role of endogenous circANRIL on apoptosis and cell proliferation.
(a) Schematic of CRISPR/Cas9-mediated knockout of ANRIL in HEK-293 cells. Genomic regions encompassing exons (Ex) 5–20 were deleted. Arrowheads indicate location and orientation of primers used for genotyping. (b) PCR genotyping. Sequencing of PCR products from Ex5–20 assay validated successful deletion in mutant cell lines (Supplementary Table 2). (c) Apoptosis and (d) proliferation in heterozygous and homozygous ANRIL knockout or control cell lines. As control, Cas9 was overexpressed without guide RNAs (gRNAs). (e) Apoptosis and (f) proliferation in homozygous knockout cells following transient re-expression of circANRIL. *P<0.05; **P<0.01; ***P<0.001. (d) Heterozygous (***P<0.001) or homozygous (#P<0.05) ANRIL knockout versus control cell lines, respectively. Comparison of multiple groups was done using analysis of variance, and Tukey was performed as post hoc test in c. Two-tailed unpaired Student's t-tests were applied in d–f. Data are given as mean±s.e.m.
Figure 4. CircANRIL does not regulate 9p21…
Figure 4. CircANRIL does not regulate 9p21 protein-coding genes and lacks miRNA sponging activity.
(a) Overexpression of circANRIL in HEK-293 cells does not modulate endogenous linANRIL RNA abundance as measured by qPCR (left panel). Expression of p16INK4a and p14ARF, encoded by CDKN2A, and p15INK4b, encoded by CDKN2B, in circANRIL-overexpressing cells (right panel) (pool of three biological replicates, quadruplicate qPCR measurements). (b) Prediction of miRNA-binding sites in circANRIL using miRanda and TargetSpy algorithms. (c) miRNA expression normalized to miR-27b as measured by qPCR (four biological replicates, quadruplicate measurements). (d) RNA immunoprecipitation (RIP) of AGO2. Analysis of precipitated RNAs: CDR1as, MALAT1, positive controls; U1, negative control (RIP was performed in a pool of three biological replicates, quadruplicate qPCR measurements). ***P<0.001. NS, not significantly different. (analysis of variance, and Tukey as post hoc test). Data are given as mean±s.e.m.
Figure 5. Identification of circANRIL- binding proteins.
Figure 5. Identification of circANRIL-binding proteins.
(a) Schematic of λN-peptide-mediated capture of circANRIL-BoxB from cellular lysates of stably overexpressing HEK-293 cell lines, and label-free mass spectrometric quantification. Experiments were performed in a pool of three biological replicates (quadruplicate measurements). (b) Quantification of RNAs by qPCR after λN-peptide capture (quadruplicate measurements). (c) Summary of circANRIL-BoxB-bound proteins according to annotated (KEGG, GO) and published functions. (d) Volcano plot of label-free quantification (LFQ) of circANRIL-BoxB-bound proteins compared with circANRIL input control. (e) RIP of PES1, NOLC1, NOP14 and mouse IgG (mIgG) control (RIP was performed in a pool of three biological replicates, quadruplicate measurements). (f) Western blot of PES1 after λN-peptide-mediated circANRIL-BoxB capture using nuclear extracts from indicated cell lines.
Figure 6. rRNA maturation defects and nucleolar…
Figure 6. rRNA maturation defects and nucleolar stress in circANRIL-overexpressing cells.
(a) Relative quantification of pre-rRNA in circANRIL- or in circHPRT1-overexpressing HEK-293 cells using isoform-specific qPCRs (RNA from a pool of three biological replicates, quadruplicate measurements). (b) Pre-rRNA levels after RNAi against circANRIL or using scrambled siRNA control (quadruplicate measurements per condition). (c) Immunofluorescent staining of PES1 in circANRIL- or in circHPRT1-overexpressing HEK-293 or empty vector control cells. Quantification of (d) nucleoli and (e) nucleolar size in circANRIL- or circHPRT1-overexpressing or empty vector control cells (*** P<0.001). Data are given as mean±s.e.m. (analysis of variance, and Tukey as post hoc test). (f) Immunofluorescent staining of p53 (red) in circANRIL- or circHPRT1-overexpressing or empty vector control cells. (g) Schematic of transcriptome and proteome analyses in circANRIL-expressing HEK-293 cells and in control cells by genome-wide expression arrays and by pulseSILAC, respectively. For results of pathway analyses and procedure, see Supplementary Tables 4–6 and Methods section, respectively. L/M/H, light/medium/heavy medium.
Figure 7. Molecular mechanism of circANRIL controlling…
Figure 7. Molecular mechanism of circANRIL controlling PES1 function.
(a) Homology of circANRIL and precursor 47S rRNA (blue boxes) determined by BLASTn algorithm. (b) Prediction of RNA–protein interaction of circANRIL with PES1 using the catRAPID algorithm. Homology regions (HR1–HR5)—predicted RNA–protein interaction motifs in circANRIL. (c) Secondary structure prediction and HR1–5 of circANRIL using the Vienna RNA package. (d) Schematic of PES1 with functional protein domains. Wild-type PES1 (PES1-WT) and two mutants lacking the 5′ (PES1Δ1–54) or the 3′ (PES1Δ446–588) lysine-rich NLSs. (e) Immunoprecipitation (IP) of PES1 isoforms from HEK-293 cells and quantification of RNA by qPCR (IP was performed in a pool of three biological replicates, quadruplicate measurements). (f) Pre-rRNA binding to PES1-WT and PES1Δ446–588 in circANRIL-overexpressing and control cells (IP was performed in a pool of three biological replicates, quadruplicate measurements). (g) Pre-rRNA and 7SL control RNA in circANRIL-expressing HEK-293 cells after transient PES1-WT or PES1Δ446–588 overexpression (pool of three biological replicates, quadruplicate measurements). (h) p53 western blot, (i), apoptosis and (j) cell proliferation in circANRIL-overexpressing HEK-293 cells with transient overexpression of PES1-WT or of PES1Δ446–588. Quadruplicate measurements per condition. *P<0.05; **P<0.01; ***P<0.001. NS, not significantly different. Analysis of variance, and Tukey as post hoc test in e,g and i. Two-tailed unpaired Student's t-tests were applied in f,j. Data are given mean±s.e.m.
Figure 8. Translation of circANRIL molecular mechanisms…
Figure 8. Translation of circANRIL molecular mechanisms in human primary cells and human cohorts.
(a) CircANRIL overexpression in primary human SMC using the green fluorescent protein (GFP)-encoding, bicistronic pBI-CMV2 vector (green; Fig. 2a) and immunofluorescent staining of PES1 (red). Quantification of nucleoli (b) and nucleolar size (c) in circANRIL-overexpressing (GFP+) SMC and circANRIL-negative (GFP−) cells. AU, arbitrary units. (d) Quantification of pre-rRNAs following circANRIL overexpression in SMC. (e) Apoptosis and (f) proliferation in SMC transfected with circANRIL. Quadruplicate measurements per condition. Correlation of endogenous circANRIL expression in human SMC with (g) apoptosis and (h) proliferation (n=5; quadruplicate measurements each). (i) Quantification of pre-rRNA following circANRIL overexpression in iPSC-derived macrophages (Supplementary Fig. 16). (j) Apoptosis in iPSC-derived macrophages transfected with circANRIL. LI, luminescence intensity. Quadruplicate measurements per condition. (k) Association of 32S rRNA expression with 9p21 A (n=49), H (n=114) and G (n=55) haplotypes in endarterectomy specimens. ***P<0.001. Two-tailed unpaired Student's t-tests were applied in b,e,f and j, and Mann–Whitney U-testing in k. Data are given mean±s.e.m.

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