Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres

Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.

. Characterization of the TERRA probe (a) Localization of probes in the cells. Fluorescence images were obtained using fixed U2OS cells expressing the TERRA probe. Alexa Fluor 488-conjugated anti-GFP antibody was used for immunostaining to label the probe with the fluorescent molecule in the cells. DNA was stained with Hoechst 33342. The probe analog without NLS was distributed in the cytoplasm (left), whereas the probe with NLS repeats localized to the nucleus (right). Scale bar, 8.0 µm. (b) Analysis of selective mPUMt binding to target RNA. Competition assays of mPUMt binding to RI-labeled 5'-UUAGGGUU-3' RNA. The competition assays were performed in the presence of increasing amounts (0-, 1-, and 10-fold molar excess) of unlabeled oligonucleotide. Four competitors were used: UUAGGGUU-RNA, Nanos Response Element (NRE) RNA, and singlestranded and double-stranded TTAGGGT-DNA. The average bound fraction for each condition was shown against the concentration of each competitor, as indicated. (c, d) EGFP reconstitution of the TERRA probe with UUAGGG repeats. (c) Fluorescence images were obtained from U2OS cells expressing iRFP-(UUAGGG) 13 (left), the TERRA probe (middle), and the merged image (right). Nucleus is indicated with white broken line. Scale bar, 5.0 µm. (d) Quantification of the number of TERRA spots in the nucleus in the cells without dox treatment (DOX(-)) and with dox treatment (DOX(+)).P-value was computed using the Student's t-test. * indicates P < 0.05. (e) Analysis of spontaneous EGFP-fragment reconstitution with probe analogs. Fluorescence images were obtained using fixed U2OS cells expressing probe analogs. The cells were immunostained with an anti-GFP antibody, which binds to full-length EGFP and the Cterminal fragment of EGFP. Alexa Fluor 561-labeled secondary antibody was used to visualize the probe localization. DNA was stained with Hoechst 33342. When the analog lacking the Nterminal fragment of EGFP and the NLS partially localized to the nucleus (upper), no fluorescence signal for EGFP was detected. Similarly, upon expression of an analog containing mPUM ACT , no fluorescence signal for EGFP was observed (bottom). Scale bar, 2.0 µm. (a) U2OS cells were transfected with siCTRL, siTERRA1, and siTERRA2. The amount of TERRA from individual subtelomeres were measured from total RNA and normalized to GAPDH mRNA. The bars represent the average values from three biological and two technical replicates for each sample. (b) Fluorescence images were obtained from U2OS cells expressing iRFP-TRF1 (telomere), the TERRA probe (TERRA), SNAP-hnRNPA1 labeled with TMR (hnRNPA1), and their merged images. The cells were transfected with siCTRL (upper), siTERRA-1 (middle), and siTERRA-2 (right). The scale bar represents 5.0 µm. (c) Quantification of the diffusion coefficients of hnRNPA1 in the cells transfected with each siRNA. n = 513 (siCTRL), 592 (siTERRA-1), 376 (siTERRA-2). (d) The number of hnRNPA1 spots observed at the telomeres in each cell condition. Mean numbers of hnRNPA1 spots per second (mean ± s.e.m) under each condition are indicated. In c and d, P-value was computed using the Student's t-test. n.s. indicates P > 0.05, * indicates P < 0.05, *** indicates P < 0.001. Time-lapse video of the U2OS cells expressing the TERRA probe and iRFP-TRF1. TERRA molecules were detected as individual fluorescent particles in the nucleus. In the raw images, background fluorescence from the out of focus area was also detected: frame rate, 100 ms/frame; scale, 4 µm.

Supplementary
Video 2. Spatiotemporal dynamics of telomere in the nucleus.
Time-lapse video of the U2OS cells expressing TERRA probe and iRFP-TRF1. iRFP-TRF1 represents telomere positions, and was observed as punctate fluorescent spots in the nucleus: frame rate, 100 ms/frame; scale, 4 µm.
Video 3. Motions of TERRA and telomere spots reconstructed as Gaussian spots.
TERRA spots and telomere spots from the Video S1 and S2 were fitted, respectively, with Gaussian spots of 200 nm diameter (TERRA, green spot) and 500 nm diameter (telomere, magenta spot): frame rate, 100 ms/frame; scale, 4 µm.
Video 4. Stationary TERRA and free-diffusing TERRA in the nucleus TERRA spots of which diffusion coefficients were lower than those of chemically fixed spots were defined as stationary TERRA (orange spots), whereas the remainder were regarded as freediffusing TERRA (cyan). The TERRA spots were overlaid with telomeres (magenta): frame rate, 100 ms/frame; scale, 4 µm.
Video 5. Formation of a TERRA-hnRNPA1 complex in the nucleus TERRA and hnRNPA1 were represented in green spots (TERRA probe, EGFP) and red spots (TMR labeled SNAP-hnRNPA1). Overlapping events exceeding 0.2 s (yellow) show a TERRA-hnRNPA1 complex formation: frame rate, 100 ms/frame; scale, 0.5 µm.