TERRA, hnRNP A1, and DNA-PKcs Interactions at Human Telomeres

Abstract

Maintenance of telomeres, repetitive elements at eukaryotic chromosomal termini, and the end-capping structure and function they provide, are imperative for preserving genome integrity and stability. The discovery that telomeres are transcribed into telomere repeat containing RNA (TERRA) has revolutionized our view of this repetitive, rather unappreciated region of the genome. We have previously shown that the non-homologous end-joining, shelterin associated DNA dependent protein kinase catalytic subunit (DNA-PKcs) participates in mammalian telomeric end-capping, exclusively at telomeres created by leading-strand synthesis. Here, we explore potential roles of DNA-PKcs and its phosphorylation target heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in the localization of TERRA at human telomeres. Evaluation of co-localized foci utilizing RNA-FISH and three-dimensional (3D) reconstruction strategies provided evidence that both inhibition of DNA-PKcs kinase activity and siRNA depletion of hnRNP A1 result in accumulation of TERRA at individual telomeres; depletion of hnRNP A1 also resulted in increased frequencies of fragile telomeres. These observations are consistent with previous demonstrations that decreased levels of the nonsense RNA-mediated decay factors SMG1 and UPF1 increase TERRA at telomeres and interfere with replication of leading-strand telomeres. We propose that hTR mediated stimulation of DNA-PKcs and subsequent phosphorylation of hnRNP A1 influences the cell cycle dependent distribution of TERRA at telomeres by contributing to the removal of TERRA from telomeres, an action important for progression of S-phase, and thereby facilitating efficient telomere replication and end-capping.

Keywords: DNA-PKcs; TERRA; hTR; hnRNP A1; strand-specificity; telomeres.

Figure 1

DNA-PKcs dependent phosphorylation of hnRNP A1 in MCF-10A and MCF-7. (A) DNA-PKcs siRNA knockdown. Real-time quantitative PCR assessment of DNA-PKcs mRNA relative expression from 24 to 120 h following transfection in mock (M), siRNA (S) treated cells, and the housekeeping gene Transferrin Receptor (TFRC). DNA-PKcs mRNA expression was normalized to TFRC levels in each sample and found to be maximally decreased at 24–48 h. DNA-PKcs protein levels were also assessed following siRNA transfection in mock (M), siRNA (S), or untreated (UT) cells over an identical time course; DNA-PKcs protein expression was normalized to the actin control. Optimal depletion of DNA-PKcs protein levels was observed at 72 h for both cell lines. An extended time course (to 240 h) monitored recovery of protein levels (not shown). (B) Overall phosphorylation status of hnRNP A1. ³²P uptake experiments followed by immunoblotting demonstrated decreased hnRNP A1 ³²P signal with DNA-PKcs depletion (siRNA) and kinase inhibition (NU7026) in MCF-7, and following DNA-PKcs siRNA silencing in MCF-10A; no decrease of hnRNP A1 ³²P signal with DNA-PKcs kinase inhibition in MCF-10A was observed.

Figure 2

hnRNP A1 siRNA knockdown in MCF-10A and MCF-7. Following hnRNP A1 siRNA transfection, cells were harvested at various times (24–228 h) and hnRNP A1 protein levels evaluated (Western blot); graphs represent hnRNP A1 levels normalized to β-tubulin. Optimal knockdown of hnRNP A1 protein (∼90%) was achieved at 72 h for both cell lines (n = 4, each examined with two immunoblot analyses).

Figure 3

TERRA co-localization at telomeres. (A) 2D analysis of TERRA and TRF2 foci. Merging of the green (TERRA) and red (TRF2/telomeres) channels denotes potentially co-localized foci as yellow signals. (B) 3D analysis of TERRA and TRF2 foci. Deconvolution and 3D reconstruction of 22 stacks per cell nuclei provided a high-resolution perspective of TERRA co-localization at individual telomeres. Navigation of the 3D image provided a defined representation of telomere location throughout the cell nucleus, as well as TERRA distribution; i.e., either bound (co-localized with telomere) or free (not co-localized with telomere). Scale bar = 8 microns.

Figure 4

Total number of co-localized TRF2/TERRA foci. Experimental groups consisted of: control (DMSO) and DNA-PK inhibitor (NU7026); control (mock) and hnRNP A1 depleted (siRNA). (A) In MCF-10A, statistically significant increases in co-localization of TERRA at telomeres (TRF2) were observed for both NU7026 and siRNA treatments (*p < 0.05). (B) In MCF-7, no statistically significant differences in co-localization of TERRA at telomeres (TRF2) were observed for either NU7026 or siRNA treatment (p > 0.05). Data are ± SEM for n = 40 TERRA positive cells.

Figure 5

Total number of TERRA foci (bound and free). Experimental groups consisted of: control (DMSO) and DNA-PK inhibitor (NU7026); control (mock) and hnRNP A1 depleted (siRNA). (A) In MCF-10A, statistically significant increases in the total number of TERRA foci were observed for both NU7026 and siRNA treatments (*p < 0.05). (B) Statistically significant increases in the total number of TERRA foci were also observed in MCF-7 for both treatments (*p < 0.05). Data are ± SEM for n = 40 TERRA positive cells.

Figure 6

Total TERRA levels. Total RNA from unsynchronized cells was extracted and TERRA probed by dot blot and normalized to GAPDH (control). RNase treatment confirmed probe specificity for RNA. Comparisons examined for statistical analyses were: DNA-PKcs kinase inhibition (NU) vs. untreated (UT) and DMSO controls; or hnRNP A1 depletion (siRNA) vs. untreated (UT2) and mock controls. (A) Total levels of TERRA in MCF-10A were not significantly affected by either treatment (p > 0.05). (B) Total levels of TERRA in MCF-7 were not significantly affected by either treatment (p > 0.05). Data are ± SEM: for n = 3.

Figure 7

Fragile telomere frequencies are increased with depletion of hnRNP A1. (A) Standard telomere FISH analysis revealed significantly elevated frequencies of fragile telomeres (arrows). (B) Two-color telomere CO-FISH was employed to evaluate strand-specificity of fragile telomeres, however no significant preference for leading- vs. lagging-strand fragile telomeres was observed (p > 0.05; data not shown). (C) Induced fragile telomere frequencies per cell and per chromosome in both MCF-10A and MCF-7 were significantly elevated following hnRNP A1 depletion (siRNA) as compared to mock controls (**p < 0.01).

Figure 8

Frequencies of TRF2, TERRA, and co-localized foci in MCF-10A and MCF-7. 3D reconstruction and analysis of co-localization facilitated distinction between bound TERRA (TERRA foci co-localized at telomeres = dark blue bar), and free TERRA (difference between total TERRA foci = gray bar and co-localized/bound TERRA = dark blue bar). Number of telomere foci are normalized to 1.00 (light blue bar). Overall relationships are maintained, but the balance of free to bound TERRA is influenced by hTR, DNA-PKcs, and hnRNP A1 interactions.

Figure 9

Speculative model of TERRA, hnRNP A1, and hTR/DNA-PKcs interactions at telomeres. The presence of “free” hTR (not associated with TERRA) stimulates DNA-PKcs phosphorylation of hnRNP A1, an event that contributes to the removal of TERRA from telomeres and may be especially important at leading-strand telomeres due to sequence complementary with TERRA. hnRNP A1 shuttling of TERRA off telomeres and out of the nucleus to the cytoplasm may aid the NMD pathway of TERRA degradation and serve to regulate TERRA levels in a cell cycle dependent manner. This supposition is supported by previous demonstrations of SMG1 stimulation and UPF1 action at leading-strand telomeres (Chawla et al., 2011). As TERRA is also complementary to hTR, when TERRA levels are low in S-phase, hnRNP A1 may also aid in the recruitment of hTR to its 3′ single-stranded overhang/substrate on newly replicated lagging-strand telomeres. When TERRA levels are high (e.g., in G1), sequestration of hTR by TERRA limits TERRA localization to telomeres, and influences the balance of free to telomere-bound TERRA.