Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication

Abstract

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5'-to-3' helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading-strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis.

Figure 1

UPF1 is present at telomeres during S and G2/M phases. (A) Chromatin immunoprecipitation (ChIP) analysis of UPF1 binding to telomeres in HeLa cells transfected with the indicated shRNA plasmids or with empty vector control plasmids (shEV). Four days after transfection, cells were harvested and ChIPs were performed with α-UPF1 rabbit polyclonal antibodies or with pre-immune (PI) serum. DNA was dot blotted and hybridized sequentially using probes detecting telomeric (Telo) or Alu repeats. (B) HeLa cells were blocked at the G1/S transition using a single aphidicolin (AΦ) block. Chromatin was prepared 0, 4 and 8 h after release (rel) from the block and ChIPs were performed with α-UPF1 or α-PCNA antibodies or with PI serum. DNA was hybridized as in (A). (C) ChIP quantifications. The amount of immunoprecipitated telomeric and Alu repeats was expressed as fractions of input DNA, after subtraction of the signal obtained for PI samples. Bars and error bars correspond to averages and s.d. from three independent experiments. P-values (two-tailed Student's t-test) are indicated for the relevant samples.

Figure 2

UPF1 binding to telomeres depends on telomere length and ATR. (A) ChIP analysis of UPF1 binding to telomeres in HeLa cells stably infected with retroviruses expressing untagged hTERT or hTERT C-terminally tagged with HA (hTERT–HA), or with empty vector (EV) retroviruses. ChIPs were performed with rabbit polyclonal antibodies raised against UPF1 and TRF1 or only with beads (b). DNA was dot blotted and hybridized sequentially using probes detecting telomeric (Telo) or Alu repeats. (B) HeLa cells were transfected with the indicated shRNA plasmids and 4 days after transfection ChIPs were performed with α-UPF1 rabbit polyclonal antibodies or with pre-immune (PI) serum. DNA was dot blotted and hybridized sequentially using probes detecting telomeric (Telo) or Alu repeats. (C) ChIP quantifications. For hTERT experiments, the amount of immunoprecipitated telomeric repeats was expressed as a fraction of input Alu DNA, after subtraction of the signal obtained for ChiPs performed only with beads. For shRNA experiments, the amounts of telomeric and Alu repeats were expressed as fractions of input DNA, after subtraction of the signal obtained for PI samples. Bars and error bars correspond to averages and s.d. from at least two independent experiments. P-values (two-tailed Student's t-test) are indicated for the relevant samples.

Figure 3

Nuclear UPF1 physically interacts with telomerase. (A) Nuclear extracts prepared from HeLa cells were immunoprecipitated using antibodies directed against the indicated proteins or only with beads (b). In all, 2% of input and 33% of immunoprecipitated material were analysed by western blot using antibodies raised against UPF1 and UPF2. The asterisk indicates a crossreacting band revealed by the α-UPF2 antibody. (B) Immunoprecipitations were performed as in (A), and 0.5% or 0.1% of input and 3.5% of immunoprecipitated material were used in TRAP assays. Numbers express TRAP activity in the different samples as fold increase over beads. (C) Myc/His-tagged UPF1 and HA-tagged hTERT plasmids were transfected in HEK 293T cells in different combinations with the corresponding empty vector (EV) plasmids. Immunoprecipitations and western blots were performed with α-myc and α-HA antibodies. In all, 3% of input and 40% of immunoprecipitated material were used for western blots. The unrelated RBM25 protein was not detected in the immunoprecipitates. A long and a short exposure (exp) are shown for α-myc western blots. (D) HEK 293T cells were transfected with the indicated plasmids and extracts were left untreated or treated with DNase I, RNase A, or both enzymes simultaneously before α-myc immunoprecipitations. Ectopically expressed UPF1 and hTERT were detected as in (C). Numbers indicate the ratio between hTERT–HA and myc/His–UPF1 signals in each IP, after normalization through the ratio measured for the untreated sample. Experiments were repeated 2–4 times and only one representative experiment is shown.

Figure 4

ATR promotes the interaction of nuclear UPF1 with active telomerase. (A, B) Nuclear extracts from HEK 293T cells left untreated or treated with caffeine were immunoprecipitated using antibodies against UPF1 or only with beads (b). TRAP experiments were performed using serial dilutions of inputs and immunoprecipitated material. (B) As in (A), except that extracts were prepared from HEK 293T cells transfected with shRNA plasmids directed against SMG1 or ATR or with empty vector plasmids (shEV). (C) Western blot analysis of UPF1, SMG1 and ATR in the indicated input or immunoprecipitated fractions. CENPA was used as a loading control. (D) Quantification of TRAP activity in the different samples after subtraction of the TRAP signal associated with the beads. Values are expressed as fold increase over untreated or shEV-transfected cells. Bars and error bars correspond to averages and s.d. from at least three independent experiments. P-values (two-tailed Student's t-test) are indicated for the relevant samples.

Figure 5

Nuclear UPF1 physically interacts with TPP1. (A) Nuclear extracts from HeLa cells were immunoprecipitated using antibodies against the indicated proteins or only with beads (b). Two independent α-TPP1 antibodies were used. In all, 3% of input and 40% of immunoprecipitated material were analysed by western blot. The asterisk indicates a crossreacting band revealed by the α-UPF2 antibody. (B) Myc/His-tagged UPF1 and Flag (FL)-tagged TPP1 plasmids were transiently transfected in HEK 293T cells in different combinations with empty vector (EV) plasmids. Immunoprecipitations and western blots were performed with α-myc and α-Flag antibodies. In all, 3% of input and 40% of immunoprecipitated material were used for western blots. The unrelated DNAPKcs protein was not detected in the immunoprecipitates. (C) HEK 293T cells were transfected with the indicated plasmids and extracts were left untreated or treated with DNase I, RNase A or both enzymes simultaneously before α-myc immunoprecipitations. Ectopically expressed UPF1 and TPP1 polypeptides were detected as in (B). Numbers indicate the ratio between FL–TPP1 and myc/His–UPF1 signals in each IP, after normalization through the ratio measured for the untreated sample. (D) Myc/His-tagged UPF1 was co-expressed in HEK 293T cells together with full-length FL–TPP1 or with a FL–TPP1 mutant lacking the N-terminal OB-fold (FL–ΔOB). Immunoprecipitations were performed with α-myc antibodies and western blot analysis was performed with α-myc and α-Flag antibodies. Experiments were repeated 2–4 times and only one representative experiment is shown.

Figure 6

The ATPase activity of UPF1 is required to sustain telomere replication. (A) Examples of telomeric DNA-FISH performed on chromosomes from HeLa cells co-transfected with shEV or shRNA plasmids against the 3′-UTR of UPF1 (shUPF1^C) and with HA-tag empty vectors (HA–EV). Telomeric sequences are in green and DAPI-stained chromosomes in red. White and black arrowheads point to telomere-free ends (TFEs) and to fragile telomeres (FTs), respectively. Enlarged chromosomes at the bottom are from shUPF1^C/HA–EV-transfected cells. (B) Quantifications of TFEs and FTs in HeLa cells transfected with the indicated plasmids. ATP^D: ATPase deficient. Frequencies are expressed as numbers of TFE and FT per chromosome. Bars and error bars are averages and s.d. from two independent experiments and n indicates the cumulative number of scored chromosomes. P-values (two-tailed Student's t-test) are indicated for the relevant samples. (C) Western blot analysis of cells used for (B). α-UPF1 (to detect endogenous and ectopically expressed UPF1), α-HA (to detect ectopically expressed UPF1 only) and α-tubulin (loading control) antibodies were used. The two lower bands detected by the α-HA antibody in UPF1wt- and UPF1ATP^D-transfected cells correspond to C-terminally truncated UPF1 proteins not detected by α-UPF1 antibody. (D) Examples of leading- and lagging-strand telomeres detected by CO-FISH analysis. Chromosomes are from HeLa cells transfected with shUPF1^C plasmids. Leading-strand telomeres are in red; lagging-strand telomeres are in green; DAPI-stained chromosomes are in blue. White and black arrowheads point to a TFE and a FT, respectively. (E) Quantifications of leading- and lagging-strand TFEs and FTs per chromosome. Bars and error bars are averages and s.d. from three independent experiments and n indicates the cumulative number of scored chromosomes. P-values (two-tailed Student's t-test) are indicated for the relevant samples.

Figure 7

Dysfunctional telomeres in cells depleted for UPF1. (A) HeLa-E1 cells transfected with the indicated shRNA plasmids were subjected to indirect immunofluorescence experiments 4 days after transfection. HRap1 is in green, γ-H2AX and RPA32 are in red and DAPI-stained DNA is in grey. Colocalization of green and red signals generates yellow signals in the merged panels (examples are indicated by the yellow arrows). The bottom panels show enlarged examples of signal colocalization from cells transfected with shUPF1^B. (B) Quantifications of hRap1 foci colocalizing with γ-H2AX or with RPA32 foci in cells transfected with the indicated shRNA plasmids. Bars and error bars represent averages and s.d. from three independent experiments where a cumulative number of 300–650 hRap1 foci were scored. P-values (two-tailed Student's t-test) are indicated for the relevant samples.

Figure 8

Speculative model on UPF1 association and function at telomeres. The white arrows indicate the direction of replication fork progression. The black arrow indicates the ATR-mediated regulation of UPF1. The black inhibitory symbol indicates the UPF1-mediated displacement of TERRA from telomeres. The dark region on TPP1 indicates the OB-fold domain that mediates the interaction with UPF1 and with telomerase. The question mark indicates one or more so far unknown factors possibly mediating the interaction between UPF1 and TPP1. See text for details.