Cross-talking noncoding RNAs contribute to cell-specific neurodegeneration in SCA7

Abstract

What causes the tissue-specific pathology of diseases resulting from mutations in housekeeping genes? Specifically, in spinocerebellar ataxia type 7 (SCA7), a neurodegenerative disorder caused by a CAG-repeat expansion in ATXN7 (which encodes an essential component of the mammalian transcription coactivation complex, STAGA), the factors underlying the characteristic progressive cerebellar and retinal degeneration in patients were unknown. We found that STAGA is required for the transcription initiation of miR-124, which in turn mediates the post-transcriptional cross-talk between lnc-SCA7, a conserved long noncoding RNA, and ATXN7 mRNA. In SCA7, mutations in ATXN7 disrupt these regulatory interactions and result in a neuron-specific increase in ATXN7 expression. Strikingly, in mice this increase is most prominent in the SCA7 disease-relevant tissues, namely the retina and cerebellum. Our results illustrate how noncoding RNA-mediated feedback regulation of a ubiquitously expressed housekeeping gene may contribute to specific neurodegeneration.

Figure 1. lnc-SCA7 regulates Atxn7 abundance

(A) Heatmap representing expression levels measured by qRT-PCR using sequence specific primers (Supplementary Table 4) [cross-threshold cycle (CT)] of lnc-SCA7, Atxn7 and Gapdh (control) (n = 3 biological replicates per tissue sample). (B) Fold difference in expression (y-axis) in N2A cells upon knockdown of lnc-SCA7 (blue) was associated with a significant reduction in Atxn7 (red) transcript abundance (C) and reduced ATXN7 protein; α-tubulin was used as loading control. (D) Over-expressing the putative 3′ noncoding region of lnc-SCA7 (lnc-SCA7-WT, dark blue), full length lnc-SCA7 (lnc-SCA7-FULL, blue) and recombinant lnc-SCA7-STOP (light blue) in N2A cells each led to significantly increased Atxn7 levels (dark red, red and pink, respectively) relative to control (white). (E) Over-expression of lnc-SCA7-WT increased ATXN7 protein; α-tubulin was used as loading control. Error bars in panels C and D, s.d.m. (n = 3 cell cultures per condition). ** p < 0.01; *** p < 0.001; Two-tailed Student’s t-test.

Figure 2. Post-transcriptional regulation by lnc-SCA7 is miR-124-mediated

Effect of lnc-SCA7’s (blue) knock-down, using si-lnc-SCA7, on Atxn7’s abundance (red) in wild-type (DTCM/D49 XY) (A) and Dcr-null (Dcr^Δ/Δ) (B) mouse embryonic stem cells relative to control (white). (C) Pairwise alignment between miR-124 and its miRNA response elements (MREs) within the 3′ UTRs of mouse lnc-SCA7 (top panels) or Atxn7 (bottom panels). The initial base of the predicted binding sites in these transcripts is noted to the left of the alignment. Consecutive identical nucleotides between miR-124 seed within lnc-SCA7 and Atxn7 are denoted by vertical lines. Difference, relative to control (white) in lnc-SCA7 (blue) and Atxn7’s (red) abundance after transfection in N2As of miR-124 mimics (D) or inhibitors (E); miR-124 levels are represented in the top-right insert (dark grey). (F) Difference, relative to control (NC, white), in luciferase activity following co-transfection, in N2As, of miR-124 mimics and luc-lnc-SCA7-WT (blue), luc-Atxn7- WT (red),luc-lnc-SCA7-MUT (light blue) or luc-Atxn7-MUT (pink). G) Changes in Atxn7 abundance (pink) following over-expression, in N2As, of lnc-SCA7-MUT (light blue), relative to control (white). (H) Changes in lnc-SCA7 abundance following over-expression of Atxn7-WT (blue) or of Atxn7-MUT (light blue), relative to control (white). Error bars in panels A,B, D-H, s.d.m. (n = 3 cell cultures per condition). ** p < 0.01; *** p < 0.001; NS p>0.05; Two-tailed Student’s t-test.

Figure 3. Transcription of miR-124 precursors is STAGA-dependent

(A) Effect in mature miR-124 levels (dark grey) of lnc-SCA7’s (blue) knockdown in N2As over a 72 hour time-course. (B) Fold enrichment in Gcn5 binding, relative to IgG control, in the promoter regions of pri-miR-124s (dark grey; Negative control, NC, white) measured using ChIP-qPCR. (C) Fold difference in normalized luciferase activity following co-transfection of empty pcDNA3.1(+) (control, white), lnc-SCA7-WT (dark grey) or lnc-SCA7-MUT (light grey) with all 3 miR-124-prom-luc reporter constructs. Error bars s.d.m. (n = 3 cell cultures per condition). ** p < 0.01; *** p < 0.001; NS p>0.05; Two-tailed Student’s t-test.

Figure 4. Crosstalking noncoding RNAs contribute to specific neurodegeneration in SCA7

(A) miR-124 (dark grey) post-transcriptionally represses and mediates crosstalk (dashed arrows) between ATXN7 (red) and lnc-SCA7 (blue) transcripts. STAGA co-activates transcription of miR-124 precursors. Red circles represent ATXN7 protein. (B) In SCA7, incorporation of mutant polyQ-ATXN7 into STAGA reduces its activity, decreases miR-124 abundance (light grey) and post-transcriptionally de-represses the miRNA’s targets.

Figure 5. Contribution of noncoding RNAs to the tissue-specific pathology of SCA7

(A) Fold difference in expression of mature miR-124 abundance (dark grey), lnc-SCA7 (blue) and ATXN7 (red) in SCA7 patient fibroblasts with 42, 49, or 55 expanded ATXN7 polyQ repeats relative to healthy control (white). (B) ChIP-qPCR revealed significantly decreased enrichment, relative to IgG control, in GCN5 binding at miR-124 promoters in SCA7^100Q/100Q mice (dark grey) relative to control animals (white). (C) Correlation between the fold difference in expression levels between lnc-SCA7 (Y-axis, blue) and Atxn7 (y-axis, red) with miR-124 (x-axis) in SCA7^266Q/5Q mice as measured using qRT-PCR and relative to matched controls, SCA7^5Q/5Q. (D and E) RNA in-situ hybridization of miR-124 and Atxn7 in the retina and cerebellum of SCA7^266Q/5Q mice and littermate SCA7^5Q/5Q controls in the retina (D; ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL)) and cerebellum (E; granule cell layer (GCL), Purkinje cell layer (PCL)). Error bars s.d.m. for 3 cell cultures per condition (A) and cerebellum tissues derived from 3 individual mice per condition (B). ** p < 0.01; *** p < 0.001; NS p>0.05; Two-tailed Student’s t-test n = 3 biological replicates per condition). Error bars represent s.d.m. **, p < 0.01; ***, p < 0.001; Student’s t-test.