RTEL1 influences the abundance and localization of TERRA RNA

Abstract

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.

Fig. 1. Domain Analysis of RTEL1.

a Schematic representation of RTEL1 proteins and constructs used in this study. The location of the RTEL1^R1264H mutation is indicated with a green star and G4 binding is illustrated. b RTEL1 interacts with itself in cells and the RTEL1^R1264H mutation disrupts interactions within the RING domain. Left, Myc immunoprecipitations from HEK293T cells co-expressing FLAG and myc-tagged RTEL1 (FL) and RTEL1^R1264H (FL ^RH) were carried out. Immunoblotting with FLAG indicates co-IP of both RTEL1 and RTEL1^R1264H. Center, immunoblotting with myc indicates co-IP of RTEL1^∆1097 (∆1097), but not RTEL1^{∆1097 R1264H} (∆1097 ^RH) with GFP-tagged RTEL1 and RTEL1^R1264H. Right, immunoblotting with FLAG shows co-IP of RTEL1^∆1097, but not RTEL1^{∆1097R1264H}. Molecular weight markers, KDa are shown. All immunoblotting experiments were repeated at least three times with similar results. c Oligomeric states of RTEL1 proteins were determined by SEC-MALS analysis of RTEL1^∆762 (blue), RTEL1^∆762R1264H (red), and RTEL1^∆762-1097 (green) at 150 and 300 mM NaCl. Normalized A280 are shown and the calculated molecular mass is shown as a line in the corresponding color across each peak with the secondary scale on the right. The expected and calculated molecular weights are shown (see also Supplementary Fig. 1).

Fig. 2. The RTEL1 C-terminal Domain Binds TERRA and other G-quadruplex Structures.

a Sequences of DNA and RNA substrates used in this study. b Nucleic acid binding of RTEL1 proteins lacking the helicase domains was monitored by fluorescence anisotropy. Bar graph shows dissociation constants (K_D) for TERRA and TERRA-MUT RNAs folded in the presence and absence of KCl and, AU-rich, and polyA RNA controls. c Increasing concentrations of the indicated oligonucleotides were added to reactions containing RTEL1 proteins and a 24mer FAM-TERRA-MUT RNA at 200 nM. Bar graphs depict apparent dissociation constants (K_i) derived by competition of the bound FAM-TERRA-MUT by the indicated oligonucleotides using fluorescence anisotropy. d K_i’s of the indicated RTEL1 deletion constructs were derived by competition studies of FAM-TERRA-MUT and indicated RTEL1 proteins at 1 µM as in panel C. No binding signal was observed for the RTEL1^∆1143 protein. All binding assays were conducted in triplicate, mean and standard deviation are shown. e RNA IP assays were done in HEK293T cells transfected with FLAG-tagged RTEL1, ∆762, TRF1 or vector only control. Precipitated nucleic acids were treated with DNAse I for 1 h at 37 °C. IP RNA was analyzed by slot blotting and detected by autoradiography using a telomeric probe. Quantitation of four independent experiments with the mean and standard deviation of relative RNA-IP normalized to the vector only control is shown. Five percent of the input is shown in the top two rows. RNAse treatment is indicated (see also Supplementary Figs. 2–7).

Fig. 3. RTEL1 Influences TERRA Levels.

a Sequence alignment of the PCR products amplified from the HEK293 cells genomic segment of RTEL1 targeted by sgRNA revealing disruption of the coding sequence (exon #2 of RTEL1). b Western blot of RTEL1 protein levels in wild type and RTEL1 knockout HEK293 cells. Molecular weight markers, KDa are shown. Immunoblotting experiments were repeated at least three times producing similar results. c Quantification of telomere loss per chromatid. Data represents the average of at least 50 metaphases as mean and standard deviation (***p < 0.001, two-sided t-test). d TERRA levels at specific chromosome ends are elevated in RTEL1 deficient cells. Bars represent the relative TERRA expression measured by qRT-PCR at 18 chromosome ends. Values are sample averages of at least four experimental repeats (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test). e TERRA levels are elevated in cells from a panel of patient derived LCL lines. TERRA levels were measured by qRT-PCR. Values are sample averages of at least three experimental repeats and p-values are listed in Supplementary Fig. 9E (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test). f TERRA levels are elevated in a fibroblast cell line derived from a HHS patient homozygous for the RTEL1^R1264H allele. TERRA levels were measured by qRT-PCR. Values are sample averages of at least three experimental repeats (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test) (see also Supplementary Fig. 9). For qRT-PCR means and standard deviations are shown.

Fig. 4. RTEL1 Influences TERRA Localization.

TERRA levels are rescued upon RTEL1 complementation. a TERRA levels for four upregulated chromosome ends in the RTEL1-KO cell line were tested in cells complemented with FLAG-RTEL1 and a catalytic site mutant FLAG-RTEL1^K48R. Bars represent the relative TERRA expression measured by qRT-PCR in three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test) and mean and standard deviation is shown. b Total subtelomeric counts determined by RNA-seq experiments in the cell lines specified in a. Mean + /− SEM is shown. c Quantification of telomere loss per chromatid in FLAG-RTEL1 and FLAG-RTEL1^K48R reconstituted cell lines by telomere FISH. d Representative telomere FISH images are shown with zoomed-in section (large white circle for zoom of small white circle). e Quantification of TERRA loss per chromatid in FLAG-RTEL1 and FLAG-RTEL1^K48R reconstituted cell lines by TERRA FISH. f Representative TERRA FISH images are shown with zoomed-in section (large white circle for zoom of small white circle). Data for both TERRA and Telomere FISH experiments represent the average of at least 50 metaphases in each of three independent experiments and three independent reconstitutions shown as the mean and standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test) (see also Supplementary Figs. 8 and 9).

Fig. 5. RNAse H1 overexpression partially phenocopies RTEL1 deficiency.

a Example chromosomes, and quantitation of TERRA free ends per chromatid in Tel FISH-positive chromatid ends. Samples were processed by sequential TERRA-Tel FISH and scored for TERRA (red) in telomeres marked by Tel FISH signal (green). Chromatid ends in at least 25 metaphases in two experiments were counted (****p < 0.0001, two-sided t-test). TERRA levels are elevated after RNAse H1 overexpression. b TERRA levels of specified chromosome ends were tested in cells transfected with RNAse H1-GFP. Bars represent the relative TERRA expression measured by qRT-PCR in three independent experiments p-values are listed in Supplementary Fig. 10B (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test). Means and standard deviation is shown. c Quantification of TERRA loss per chromatid in cells transfected with RNAse H1-GFP by TERRA FISH (22 days in selection). d Quantification of telomere loss per chromatid in cells transfected with RNAse H1-GFP by telomere FISH (22 days in selection). e Quantification of telomere loss per chromatid in cells transfected with RNAse H1-GFP by telomere FISH (52 days in selection). Three independent cell lines complemented with RTEL1 were averaged for analysis where specified. FISH experiments represent the average of at least 50 metaphases in each of 2 independent experiments and three independent reconstitutions shown as the mean and standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t-test) (see also Supplementary Fig. 10).

Fig. 6. RTEL1 Influences the Abundance and Localization of the Telomeric RNA TERRA.

The formation and maintenance of the TERRA R-loop at the telomere influences TERRA transcription and promotes telomere stability. We propose RTEL1 would be involved in either facilitating the creation or contributing to the maintenance of the TERRA-containing telomeric R-loop. Once established, the TERRA R-loop would limit further transcription from R-loop containing chromosome ends.