Telomeric RNAs are essential to maintain telomeres

Telomeres are transcribed generating long non-coding RNAs known as TERRA. Deciphering the role of TERRA has been one of the unsolved issues of telomere biology in the past decade. This has been, in part, due to lack of knowledge on the TERRA loci, thus preventing functional genetic studies. Here, we describe that long non-coding RNAs with TERRA features are transcribed from the human 20q and Xp subtelomeres. Deletion of the 20q locus by using the CRISPR-Cas9 technology causes a dramatic decrease in TERRA levels, while deletion of the Xp locus does not result in decreased TERRA levels. Strikingly, 20q-TERRA ablation leads to dramatic loss of telomere sequences and the induction of a massive DNA damage response. These findings identify chromosome 20q as a main TERRA locus in human cells and represent the first demonstration in any organism of the essential role of TERRA in the maintenance of telomeres.

RNase. Scale bar: 10 µm. (C) Northern blotting using 32 P-dCTP-labelled probes on total RNA from HCT116 cells DKO (for the DNMT1 and 3b genes) or HCT116 wild-type, HeLa and U2OS treated and untreated with 5′Azacytidin (Aza) for 72 hours. The RNA was used to prepare two identical membranes, in one of them the TERRA probe was hybridized first and, upon stripping, the WASH probe. In the other membrane, the DDX probe was hybridized first and then the TAR1 probe. 18S was included as a loading control. The expected size for DDX transcripts is 1.4-1.6kb and for WASH 1.3-1.8Kb. For TAR1 region, no annotated transcripts have been described so far. *Unspecific band due to cross-hybridization with rRNA 18S and 28S. (D) The RNA extracted in (B) was also used for RNA dot-blot to detect either TERRA-UUAGGG track, DDX11L or WASH transcripts; 18S serves as loading control. All the hybridizations were performed on the same membrane one after the other upon stripping.
(Graphs) Dot-blot quantification normalized by 18S (mean values±s.e.m., n=3 biological replicates). The Student´s t-test was used for the statistical analysis of the comparison between untreated and Aza-treated cells. One-way Anova with Tukey post test was used for the statistical analysis of the comparison of one cell line with the others (*p < 0.05, **p < 0.01 and ***p < 0.001).

Supplementary Figure 2. Different combinations of gRNAs are able to delete the 20q and
Xp loci in different cell lines. IMR90 non-transformed human fibroblasts, HeLa, HCT166 and U2OS cell lines were transfected with different combinations of Cas9-GFP-plasmids containing two different gRNAs to delete 10Kb of the 20q and XpYp region enriched in TERRA transcripts. Two-days upon transfection total DNA was isolated from the different cells lines and PCR using specifics primers to detect the deletion was performed and visualized in ethidium bromide gels. The individual gRNAs used in each combination (C1, C2, C3 and C4) (gRNA mix) are shown below. The primers for the gRNAs synthesis can be found in the  TIDE (Tracking of Indels by DEcomposition) analysis to study the four top of targets for the gRNAs (START2 and END1) used to generate the 20q locus deletion. The analysis was performed with DNA pools from either wild type cells or cells bearing the 20q deletion, named C3 (from which the clones A2, B4 and C4 were obtained from). A DNA that did not undergo CRISPR treatment was used as reference. (B) TIDE analysis for the off-targets 3, gRNA END1

Supplementary
using DNAs from the 20q-KO clones A2, B4 and C4 and from cells that did not undergo CRSIPR treatment. A DNA from other set of cells that did not undergo CRISPR treatment was used as reference (C) T7 endonuclease assay was performed on DNAs from the regions of interest (off-target2/START2, off-target 3/END1 and off-target 4/END1) obtained from pool of cells bearing the 20q deletion (C3) or from the 20q-KO clones. Two identical DNAs except for a C to G mutation in one of them (named C and G) were used as positive control. The ethidium bromide gels shows the different DNAs upon T7 endonuclease digestion.

Supplementary Figure 6 Detection of shelterins in 20q-TERRA KO cells. (A)
Quantification of immunoprecipitated telomeric repeats upon TRF2-or TRF1-ChIP. Values were obtained after normalization to telomeric DNA input and corrected by the differences in the telomere length between the clones and wild-type cells (see Figure 4). Error bars correspond to the 2-3 independent experiments (mean values± s.e.m., n=2-3 independent experiments). One-way Anova with Tukey post test was used for statistical analysis (**p < 0.01). (B) Representative images of TRF2 and TRF1 immunoflorescence (green) in wild-type cells and 20q-KO TERRA cells (clone C4). Merge images with DAPI are also shown (Graphs).
Quantification of the mean fluorescence intensity of TRF1 and TRF2 is represented (mean values± s.e.m., n=3 independent experiments). The Student´s t-test was used for statistical analysis (*p < 0.05). Scale bar: 10 µm.