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The Ro60 autoantigen binds endogenous retroelements and regulates inflammatory gene expression

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Science  23 Oct 2015:
Vol. 350, Issue 6259, pp. 455-459
DOI: 10.1126/science.aac7442

An Aluring new autoantibody target

Autoimmunity is the immune system's ultimate act of betrayal. Cells designed to protect against invading microbes suddenly target the host instead. In the autoimmune disease systemic lupus erythematosus, antibodies target DNA and host proteins, including the RNA binding protein Ro60. Hung et al. discovered that Ro60 bound to endogenous Alu retroelements. They detected antibody-Ro60-Alu RNA immune complexes in the blood of individuals with lupus and an enrichment of Alu transcripts. Ro60 bound to Alu probably primes RNA-binding innate immune receptors within B cells, leading these cells to make antibodies that target Ro60-Alu RNA and drive disease-causing inflammation.

Science, this issue p. 455

Abstract

Autoantibodies target the RNA binding protein Ro60 in systemic lupus erythematosus (SLE) and Sjögren’s syndrome. However, it is unclear whether Ro60 and its associated RNAs contribute to disease pathogenesis. We catalogued the Ro60-associated RNAs in human cell lines and found that among other RNAs, Ro60 bound an RNA motif derived from endogenous Alu retroelements. Alu transcripts were induced by type I interferon and stimulated proinflammatory cytokine secretion by human peripheral blood cells. Ro60 deletion resulted in enhanced expression of Alu RNAs and interferon-regulated genes. Anti-Ro60–positive SLE immune complexes contained Alu RNAs, and Alu transcripts were up-regulated in SLE whole blood samples relative to controls. These findings establish a link among the lupus autoantigen Ro60, Alu retroelements, and type I interferon.

Autoantibodies to the RNA binding protein Ro60 (also called SSA) are present in individuals with systemic lupus erythematosus (SLE) (1), Sjögren’s syndrome (2), neonatal lupus with heart block (35), and other autoimmune disorders (6). SLE subjects with anti-Ro60 antibodies have an increased prevalence of skin disease, photosensitivity, and nephritis, along with elevated expression of interferon (IFN)–inducible genes in immune cells and tissues (7, 8). Ro60, encoded by TROVE2, is a component of a ribonucleoprotein complex comprising the E3 ubiquitin ligase Ro52 (9), the polymerase III (Pol III) transcriptional terminator La/SSB, and short noncoding Y RNAs (HY1-4) (10). Ro60 has a specific RNA recognition domain for Y RNAs but binds additional, possibly misfolded, RNAs through a separate cavity (11). The identity of these other RNA species and their relevance to autoimmune disease is unclear.

To identify RNAs bound to Ro60, we performed iCLIP (individual nucleotide cross-linking and immunoprecipitation) (1214) followed by high-throughput sequencing in two type I interferon–stimulated ENCODE cell lines: GM12878, an Epstein-Barr virus–transformed B lymphocyte cell line, and K562, an erythromyeloblastoid leukemia cell line (www.genome.gov/encode/) (fig. S1). Sequencing reads were aligned to the human reference genome (hg19), generating 970,667 and 1,090,671 alignments for GM12878 and K562, respectively. This analysis identified 2445 Ro60-bound sites in GM12878 and 8273 sites in K562.

Annotation of all peaks to known genomic features revealed that approximately two-thirds of the iCLIP tags localized to introns in both cell lines (Fig. 1A). As expected, Y RNAs constituted a substantial fraction of iCLIP tags in both lines (Fig. 1B). Surprisingly, iCLIP tags within Alu SINEs (short interspersed elements), a common repetitive transposable element (~1.1 M) in the human genome (15), were also represented (Fig. 1, B and C). Additional lower-frequency tags included 5′ and 3′ untranslated regions, LINEs (long interspersed elements), tRNAs, rRNAs, and simple repeats (fig. S1). Most of the Ro60 iCLIP peaks identified in GM12878 (1465/2445, 60%) were also found in K562 (Fig. 1D and table S1), indicating concordance of iCLIP results between the two cell lines.

Fig. 1 iCLIP identifies RNAs bound by Ro60.

(A) Pie graph showing annotation of Ro60 iCLIP peaks to gene structures in IFN-α–treated (6 hours) K562 and GM12878 cell lines. (B) Pie graph showing annotation of iCLIP peaks to Repeatmasker elements. (C) Genome browser snapshot of a representative Alu iCLIP peak; y axis indicates read counts. (D) Venn diagram representation of overlap in Ro60 iCLIP peaks between K562 and GM12878. P < 0.001, hypergeometric test.

Motifs enriched within shared iCLIP peaks included Y RNA consensus sequences, as well as a U-rich motif that corresponds to the 3′ antisense strand of an Alu element (P < 10−1800) (Fig. 2A and fig. S2). The interaction between Ro60 and Alu RNA was confirmed using both RNA immunoprecipitation (RIP)–polymerase chain reaction (PCR) (Fig. 2B) and RIP-seq (fig. S3). RIP-PCR over a 12-hour time course of IFN-α treatment of GM12878 showed binding of HY4 RNA to Ro60 at baseline, and this association peaked ~5 hours after IFN-α treatment (Fig. 2B). Alu RNAs were also bound to Ro60 and showed complex kinetics of association after IFN-α treatment, with the highest levels measured at 12 hours.

Fig. 2 Ro60 binds an RNA motif derived from Alu elements.

(A) Top four RNA motifs (14) identified from the Ro60 iCLIP peaks shared between K562 and GM12878. T = U in RNA. (B) RIP-PCR for Alu or HY4 RNA in Ro60 or IgG immunoprecipitates across a time course of IFN-α treatment in GM12878 cells. Error bars represent SD of three technical replicates; data shown are representative of three experiments. (C) EMSA of Alu RNA motif [oligo b from (D)] with indicated concentrations of recombinant Ro60 protein. (D) EMSA of Alu motif variants with recombinant Ro60. a, PolyU sequence alone; b, representative Alu motif; bas, antisense of b; c, deleted polyU; d, partial 5′ and 3′ truncations; e, partial 3′ truncation. Asterisk indicates RNA/Ro60 complex. RNA sequences depicted in bottom panel. See also fig. S4.

We next used an electromobility shift assay (EMSA) to test the binding of radiolabeled Alu motif RNA oligonucleotides to purified recombinant Ro60 protein. Ro60 binding was observed with the full-length Alu motif (Fig. 2, C and D, oligo b), and an antisense version of the full-length motif (Fig. 2D, oligo bAS). Reduced binding to Ro60 (oligos d and e), or no appreciable binding (oligos a and c), was observed for various truncated Alu motifs (Fig. 2D). Several sequence mutants that abrogated the predicted secondary structure of the Alu motif did not affect binding to Ro60; however, mutations to the polyU tract disrupted the interaction (fig. S4).

To further explore the functional role of Ro60, we used zinc finger nuclease technology to generate three GM12878 Ro60 knockout (KO) cell lines, each containing unique biallelic frameshift mutations within exon 2 of Ro60/TROVE2 (fig. S5). The KO lines contained undetectable levels of Ro60 protein (Fig. 3A) and showed markedly reduced retrieval of Alu and Y RNA by Ro60 RIP-PCR (fig. S6). Surprisingly, RNA-seq at baseline and 6 hours after treatment with IFN-α demonstrated that many of the genes altered by IFN-α treatment in the parental cells (lanes 9 and 10) were dysregulated in the Ro60 KOs in the absence of treatment (Fig. 3B, lanes 3 to 5). Gene expression correlated significantly between the unstimulated Ro60 KO lines and the IFN-α–treated parental cell line (Fig. 3C) (R = 0.64, n = 4520, P < 0.0001). Genes differentially expressed in the Ro60 KO lines at rest were enriched for a number of immune-related GO terms and Kegg pathways (fig. S7), suggesting aberrant activation of inflammatory pathways in the absence of Ro60 (see also table S2) (14).

Fig. 3 Loss of Ro60 in GM12878 cells activates inflammatory gene expression and Alu RNA expression.

(A) Western analysis of Ro60, Ro52, La, and tubulin in three independent GM12878 Ro60 KO clones. (B) RNA-seq heat map of 1698 genes induced or repressed by a factor of >2 upon 6 hours of IFN-α in parental GM12878 cells. Unsupervised clustering of both genes and conditions. Lanes 1 and 2: Parental cells at baseline, biologic replicates. Lane 3: KO1 at baseline. Lane 4: KO2 at baseline. Lane 5: KO3 at baseline. Lane 6: KO1 + IFN-α. Lane 7: KO2 + IFN-α. Lane 8: KO3 + IFN-α. Lanes 9 and 10: Parental cell line + IFN-α, biologic replicates. See fig. S7 for global correlation of gene expression between clones. (C) Scatterplot of gene expression changes [log2(relative change)] in Ro60 null clones (x axis) or wild-type parental cells treated with IFN-α (y axis) compared to baseline wild-type cells. Data represent all genes with induction or repression by a factor of ≥1.5; n = 4520, P = 2.2 × 10–16. R = Pearson product-moment correlation. (D) Western blot of whole-cell lysates at indicated time points after IFN-α treatment of the parental cell line. (E and F) RT-PCR of HY4 RNA (E) or Alu RNA (F) at indicated time points after IFN-α treatment of the parental cell line. Expression values normalized to GAPDH. P values of (E): KO1 versus WT (P = 0.015), KO2 versus WT (P = 0.001), KO3 versus WT (P = 0.001); Student’s t test. (G) Quantitation of Alu reads as a percent of all RNA-seq reads from IFN-α–treated (6 hours) wild-type (biologic replicates, n = 2) or Ro60 null clone (n = 3) libraries. Error bars represent SD. (H) Enzyme-linked immunosorbent assay (ELISA) of culture supernatants of human PBMCs 15 hours after N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) transfection of the indicated RNAs (see Fig. 2D for RNA sequences). Error bars represent SD of three technical replicates from a representative experiment (1 of 3). (I) ELISA of culture supernatants of human PBMCs 15 hours after DOTAP cotransfection of the indicated RNAs with TLR7/9 inhibitory oligo IRS954 or a control oligo. Error bars represent technical replicates from a representative experiment (1 of 4).

We next performed a time course of IFN-α treatment to determine whether the widespread changes in gene expression included alterations in Ro60-bound RNAs. In the parental cell line, there were minimal changes in the protein levels of Ro60 during the 12-hour experiment (Fig. 3D). At all time points, Y RNAs were expressed at reduced levels in the Ro60 null cells relative to parental cells (Fig. 3E and fig. S7), whereas total Alu RNAs were found at significantly higher levels in the null cells (Fig. 3F). We confirmed these differences by quantitation of Alu reads from the RNA-seq data (Fig. 3G). Together, these findings demonstrate elevation of Alu expression and activation of IFN-induced genes in the absence of Ro60. A possible interpretation of these data is that aberrant expression of endogenous Alu RNAs stimulates intracellular RNA sensors to induce inflammatory responses.

To test this idea, we transfected synthesized RNAs into peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. The Alu motif RNAs potently stimulated secretion of cytokines, including IFN-α, interleukin (IL)–6, and tumor necrosis factor (TNF)–α (Fig. 3H), consistent with a previous report (16). Cytokine induction by the Alu RNAs was blocked by co-incubation of the transfected cells with chloroquine, a molecular agent that disrupts signaling of endosome-localized Toll-like receptors (TLRs) 3, 7, and 8, all of which recognize RNA (17, 18), or by cotransfection of a TLR7/9 antagonist, phosphorothioated oligo IRS954 (19, 20) (Fig. 3I). Co-incubation of the transfected PBMCs with BX795, an inhibitor of TBK/IKKe (21) that mediates signaling downstream of cytosolic RNA receptors RIG-I, MDA-5, and TLR3, had no effect on Alu-mediated cytokine production (fig. S8).

Truncated Alu motifs (see Fig. 2D) elicited submaximal cytokine responses. Y RNAs showed no ability to elicit cytokine production in this system. Alu motif sequence variants that retained Ro60 binding in vitro (fig. S4) also elicited cytokine responses comparable to the wild type (fig. S9). We conclude that cytokine secretion in Alu motif–transfected human PBMCs is TLR7-dependent.

Next, we purified SLE serum immunoglobulin G (IgG) using Protein G beads from subjects representing a spectrum of anti-Ro autoantibodies (fig. S10) and performed immunoprecipitations from GM12878 and K562 cell lysates. By Western blot, only sera II and IV immunoprecipitated Ro60 (Fig. 4A). Sera II and IV also precipitated Y RNA and POLG RNA (fig. S10), two of the prominent peaks identified in the Ro60 iCLIP experiments. Using primers spanning the 284–base pair consensus Alu sequence (Fig. 4B), RT-PCR showed that endogenous SLE IgGs also immunoprecipitated Alu transcripts (Fig. 4C). Parallel experiments in the Ro60 null cell lines showed reduced immunoprecipitation of Alu and Y RNAs (fig. S11). We found that Alu RNAs were enriched in primary serum IgG immune complexes from anti-Ro60–positive SLE patients (Fig. 4D), which suggests that circulating anti-Ro60 autoantibodies in SLE subjects are preloaded with Alu transcripts.

Fig. 4 Alu RNAs in Ro60-positive SLE immune complexes and SLE whole blood.

(A) Immunoprecipitation from IFN-α–treated (6 hours) K562 or GM12878 cell lysates using SLE patient IgGs followed by Western blot of Ro52 or Ro60. Input lane represents 10% total lysate. (B) PCR primer map of Alu elements with respect to the consensus Alu sequence and the identified Ro60 Alu motif from Fig. 2A (purple arrow represents orientation of the motif). (C) SLE IgG immunoprecipitates from IFN-α–treated K562 lysates in (A) were subjected to RT-PCR of Alu RNA using the primers in (B). Error bars represent SD of representative experiment (of 3). (D) RT-PCR of cell-free serum IgGs purified using Protein G beads from 50 μl of SLE sera (I-V). Values represent relative change with respect to patient I IgG. Preexisting Alu RNAs contributed negligibly to the total Alu RNAs retrieved from cell lysates in part (C) (fig. S11C). (E) Percent of whole blood RNA-seq Alu reads in control or SLE subjects. P value calculated using Student’s t test. Error bars represent SD. (F) Heat map of the top 1641 Alu elements expressed in SLE or control subject blood. Unsupervised hierarchical clustering of Alus and subjects. Bars at the top represent anti-Ro titers (blue) or Interferon Signature Metric (ISM) scores (red = ISM high, pink = ISM low). (G and H) Alu score = mean expression of top SLE Alu RNAs (14). (G) Alu scores binned by ISM status; (H) Alu scores binned by anti-Ro status. (I) Distribution of gene expression changes in IFN-α–treated (6 hours) PBMCs relative to controls; expression is the average of three donors. For Alu density calculations, see (14). P < 0.001 (Q4 versus Q3).

Finally, we tested the levels of Alu transcripts in blood cells of SLE patients and controls (22) using RNA-seq. (99 active SLE, 18 healthy controls; fig. S12). RNA-seq reads mapping to Alu elements were found at significantly higher levels in SLE subjects than in controls (P = 6.5 × 10–6) (Fig. 4E). Hierarchical clustering of the most highly expressed Alu RNAs (fig. S13) segregated Interferon Signature Metric (ISM)–high SLE subjects from control and ISM-low patients (Fig. 4F). Alu scores (14) were higher in ISM-low SLE subjects than in controls and were highest in ISM-high SLE subjects (Fig. 4G). Alu scores were also significantly elevated in anti-Ro autoantibody–positive SLE subjects relative to SLE subjects lacking anti-Ro (P = 0.01) (Fig. 4H). A subset of ISM-high/Alu score–high SLE subjects lacked anti-Ro antibodies, indicating that there are other mechanisms for induction of IFN and Alu RNA in SLE subjects, likely including autoantibodies to other RNA binding proteins (23).

Endogenous retroelements were recently shown to activate humoral immune responses in mice (24), and aberrant accumulation of retroelements contributes to two human genetic diseases with features of SLE: Aicardi-Goutieres syndrome and familial chiblain lupus (25). Our investigation of the Alu RNAs bound to Ro60 supports the hypothesis that retroelements contribute to human autoimmunity. Our findings suggest a model that links Ro60 autoantibody responses and Alu retroelement stimulation of intracellular RNA sensors, including TLR7, with development of SLE, Sjögren’s syndrome, and congenital heart disease in the offspring of anti-Ro60 mothers (4) (see fig. S14). We speculate that IFN-stimulated modifications to Alu RNA, such as N6-methyladenosine (m6A), may mediate this interaction (fig. S14B). These findings also provide new insights into the lupus-like disorder described in Ro60 knockout mice (26), suggesting that autoimmunity in this model may be mediated by dysregulation of murine retroelements.

Finally, this work may help to explain the maintenance of Alu sequences in the genome over the course of primate evolution. There are more than 1 million copies of the Alu retroelement in the human genome, and it has been postulated that the retention of Alu sequences in gene-rich regions and their transcription in response to stress might provide an evolutionary advantage (27). When we binned all coding genes in quartiles from high to low density of the Alu motif and plotted gene expression changes upon IFN-α treatment, we observed that genes with higher densities of the Alu motif showed increased IFN-inducible gene expression (P < 0.001; Fig. 4I and fig. S14A). These data support a model where the transcription of Alu-containing genes upon pathogen infection up-regulates endogenous Alu RNA “adjuvants,” which serve to amplify immune responses via stimulation of intracellular RNA sensors, possibly by temporarily overwhelming the ability of endogenous Ro60 to target these RNAs for destruction. Thus, Alu sequences may have persisted in the genome by virtue of their beneficial effects for defense against infectious agents, while at the same time predisposing to anti-Ro60 autoantibody production in the setting of systemic autoimmunity.

Supplementary Materials

www.sciencemag.org/content/350/6259/455/suppl/DC1

Materials and Methods

Figs. S1 to S14

Data Tables S1 and S2

References and Notes

  1. See supplementary materials on Science Online.
  2. Acknowledgments: We thank J. Lowe, E. Brown and V. Dixit for their critical review of the manuscript. The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. A material transfer agreement is required for access to the GM12878 Ro60 KO cell lines. Sequencing data are available at NCBI GEO, accession number GSE72420. Supported by an Alfred P. Sloan research fellowship and NIH grants HG004659, NS075449, and HG007005 (G.W.Y.) and by NSF graduate research fellowship grant DGE-1144086 (G.A.P.).
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