hnrnp D/AUF1 Rabbit IgG hnrnp M

Transcription

1 Mouse IgG Goat IgG Rabbit IgG Mouse IgG hnrnp F Goat IgG Mouse IgG Kb Supplementary Figure S1. In vivo binding of TERRA-bound RBPs to target RNAs. Immunoprecipitation (IP) assay using 3 mg of nuclear extracts prepared from primary MEFs and 3 µg of either RBPs antibodies or IgG1, under conditions that preserved mrnp complexes. RNAs isolated from IP material were separated in an RNA gel and detected by ethidium bromide staining. RNA signals were found on the,, A2, D, M and F lanes. -IP is shown twice because the signal of the immunoprecipitated RNAs on the left panel (used for Fig. 2b) is hidden by the strong signal on the -IP lane. Size is shown in base pairs.

2 C F M A1 H A2 D/AUF: sirna : RNase H 6 4 with polya w/o polya S Supplementary Figure S2. TERRA polyadenylation upon downregulation of TERRA-bound RBPs. Two days after sirna transfection, total RNA from primary MEFs was harvested, annealed with oligodt and digested with RNase H. Changes in TERRA migration were monitored by northern blot using a 32P-dCTP labelled 1.6kb telomeric probe; hybridization of 18S was included as a loading. sirnas used were: (C), (A1), (H), hnrnp F (F), (M), (A2), and (D/AUF1). Size is shown in base pairs.

3 a b Untr ActD Dapi Merge Percent mrna remaining 1 t 1/2 (GAPDH)= 7.7 hrs t 1/2 (c-fos)= 1.51 hrs c-fos GAPDH Supplementalry Figure S3. translocates into the cytoplasm upon Actinomycin D treatment and c- fos and GAPDH mrna decay. a. staining upon Actinomycin incubation (5 µg/ml) for 4 hrs. Dapi was used for nuclear staining. Scale bars represent 5 µm. b. c-fos and GAPDH half-life was measured by incubating pmefs with actinomycin D, extracting total RNA at the times shown, and measuring mrna levels by RT-PCR. The data were normalized to 18S rrna levels and represented as a percentage of the mrna levels measured at time, before adding actinomycin D, using a semilogarithmic scale. The half-lives (t1/2) were calculated as the time required for the mrna to decrease to 5% of its initial abundance (discontinuous horizontal line). The mean and standard error of the mean (s.e.m) of the percentage from three independent experiments are represented

4 sirna Control C : ActD (hrs) TERRA t 1/2 (A1 sirna)>8 h t 1/2 (A2 sirna)>8 h Control Control TERRA 18S 18S t 1/2 (H sirna)>8 h t 1/2 (M sirna)>8 h t 1/2 (AUF sirna)>8 h Supplementary Figure S4. TERRA stability upon downregulation of TERRA-bound RBPs. TERRA half-life after silencing of TERRA-bound RBPs was measured by incubating cells with actinomycin D, extracting total RNA at the times shown, and measuring TERRA levels by dot-blot analysis. The data were normalized to 18S rrna levels and represented as a percentage of the TERRA levels measured at time, before adding actinomycin D, using a semilogarithmic scale. The half-lives (t1/2) were calculated as the time required for TERRA to decrease to 5% of its initial abundance (discontinuous horizontal line). The mean and standard error of the mean (s.e.m) of the percentage from three independent experiments are represented

5 C A1 H F M A2 D/AUF: sirnas Nuclear Cytoplasmic TERRA Nuclear Cytoplasmic GAPDH Supplementary Figure S5. Nuclear and cytoplasmic TERRA upon TERRA-bound RBP downregulation. Two days after sirna transfection, nuclear and cytoplasmic fractions from primary MEFs were prepared and the RNA was isolated with Trizol LS (Invitrogen). TERRA signal was detected by dot-blot using a 32P-dCTP labelled 1.6kb telomeric probe; hybridization of GAPDH was included as a loading. sirnas used were: (C), (A1), (H), hnrnp F (F), (M), (A2B1), and (D/AUF1)

6 a Control A1 H F M A2B1 D/AUF1 : sirna Merge Rap1 TRF1 b c d TRF1 foci/nucleus Mean = n=4 nuclei Mean = Mean = n=21 nuclei n=23 nuclei Mean = n=26 nulcei Mean = Mean = n=22 nuclei n=38 nuclei Mean = n=24 nuclei Rap1 foci/nucleus Mean = n=4 nuclei Mean = n=38 nuclei Mean = Mean = n=21 nuclei n=23 nuclei Mean = Mean = n=26 nulcei n=22 nuclei Mean = n=24 nuclei C A1 H F M A2B1 D/AUF1 C A1 H F M A2B1 D/AUF1 TRF1/Rap1 colocalization/nucleus Mean = n=4 nuclei Mean = Mean = n=21 nuclei n=23 nuclei Mean = n=26 nulcei Mean = Mean = n=22 nuclei n=38 nuclei Mean = n=24 nuclei C A1 H F M A2B1 D/AUF1 Supplementary Figure S6. Impact of TERRA-bound RBPs on shelterin proteins. a. Representative images of TRF1 (green), and of Rap1 (red) fluorescence, and of the merged images. Co-localization events are detected by yellow fluorescence. Scale bars represent 5 µm. b. Quantification of TRF1 foci/nucleus, c. Rap1 foci/nucleus and d. colocalizing RAP1/TRF1 foci/nucleus. Mean fluorescence value (red bar) and standard error of the mean (s.e.m.) are represented. n=total number of nuclei analyzed per condition. A total of three independent sirna transfection experiments were used for the analysis. The Student s t-test was used for statistical analysis (p<.5,p<.1, and p<.1). sirnas used were: (C), (A1), (H), hnrnp F (F), (M), (A2B1), and hnrnp D/AUF1 (D/AUF1).