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Fig. S1. The GAA-repeat-containing RNA forms intranuclear foci. (A) Schematic map of the cDNA (AK082015) and probes that were used for RNA-FISH analysis. Fragment-2 probe contains the GAA.TTC tandem repeat region (red). (B) RNA-FISH using F1-R1 (a, d), F2-R2 (b,e) and F3-R3 (c,f) probes in NIH-3T3 cells revealed that only the F2-R2 probe hybridized to RNA distributed in the form of punctate nuclear foci (arrows). (C) RNA-FISH using fragment-1 (a,d), fragment-2 (b,e) and fragment-3 (c,f) probes in NIH-3T3 cells revealed that only the fragment-2 probe hybridized to RNA distributed in the form of punctate nuclear foci (arrows). The DNA was stained with DAPI (blue). Scale bar: 10 µm.
Fig. S2. GRC-RNA foci are not degraded by double-stranded-specific RNase treatment but are sensitive to RNase H treatment post-hybridization with a DNA oligonucleotide probe. GRC-RNA foci (green) were resistant to double-stranded RNases (RNase VI; a,c and RNase III; d,f). Note that the Neat1 RNA (red) was sensitive to both RNase VI (b,c) and III (e,f) treatment. Co-RNA-FISH in NIH-3T3 cells using GRC-RNA (green) and Neat1 RNA probes (red) revealed that both GRC-RNA and Neat1 RNA foci were sensitive to RNase-H-mediated degradation only if the cells were incubated with RNase H after the hybridization (j-l) and not prior to hybridization (g-i) with DNA probes. DNA was counterstained with DAPI. Scale bar:10 µm.
Fig. S3. The GAA-antisense DNA oligonucleotide transfection in NIH-3T3 cells results in the degradation of GRC-RNA nuclear and cytoplasmic foci. RNA-FISH for GRC-RNA in NIH-3T3 cells that were transfected with phosphorothioate-modified scrambled DNA oligonucleotide (control; a,b), GAA-sense DNA oligonucleotide (control; c,d) and GAA-antisense DNA oligonucleotide (e,f) revealed that only GAA-antisense oligonucleotide-transfected cells lost both nuclear and cytoplasmic GRC-RNA foci (compare e with a and c). DNA is counterstained with DAPI. Scale bar: 10 µm.
Fig. S4. GRC-RNA foci associate with GAA.TTC-repeat DNA elements. RNA-FISH using a (TTC)15 (red; a,e,i,m) probe followed by DNA-FISH using a (GAA)15 probe (green; b,f,j,n) revealed association of GRC-RNA with GAA.TTC-repeat DNA elements in the genome. Merge is shown in c, g, k and o. Note that in a-d, the arrows represent GRC-RNA foci that colocalize with GAA.TTC-repeats, open circles represent the GRC-RNA foci that do not associate with GAA.TTC repeats and arrowheads represent the GAA.TTC-repeat DNA regions that do not associate with GRC-RNA foci. a-d are also shown in Fig. 3B. The DNA is stained with DAPI. Scale bar: 10 µm.
Fig. S5. GRC-RNAs localize to a novel nuclear domain. Dual localization of GRC-RNA (red; a,e,i,m,q,u) with markers of known subnuclear domains in NIH-3T3 cells did not show any colocalization (b, Ana-C Ab for centromere; f, TERRA nrRNA-FISH for telomere; j, Neat1 nrRNA for paraspeckle; n, SF2/ASF Ab for nuclear speckles; r, Coilin Ab for Cajal bodies; and v, CFP-PML for PML bodies). Merge is shown in c, g, k, o, s and w. Chromatin was counterstained with DAPI (blue; d,h,l,p,t,x). Scale bar: 10 µm.
Fig. S6. GRC-RNA foci in WT-MEFs are sensitive to prolonged incubation of α-amanitin. Co-RNA FISH in WT-MEFs using probes to GRC-RNA (green; a,d,g) and Neat1 (b,e,h) revealed that prolonged incubation (14 hours) of cells with RNA polymerase II inhibitor α-amanitin resulted in the degradation of GRC-RNA foci (compare g with a). Note that the Neat1 RNA foci were lost within 6 hours of α-amanitin treatment (e). Merge is shown in c, f and l. The DNA is counterstained with DAPI (blue). Scale bar: 10 µm.
Fig. S7. The GRC-RNA localized to the midbody in NIH-3T3 cells. Co-RNA FISH in NIH-3T3 telophase (a-c) and early G1 cells (d-f) using probes to GRC-RNA (green; a,d) and Neat1 (red; b,e) revealed that only GRC-RNA localized to the midbody. The arrows designate the position of the midbody in telophase and early G1 cells. The DNA is counterstained with DAPI (blue). Scale bar: 10 µm.