The molecular mechanism for TERRA recruitment and annealing to telomeres

Abstract

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere.

Graphical Abstract

Figure 1.

TERRA association with telomeric duplex DNA is enhanced by sub-telomeric sequence. DNA construct: (A) Cy5 labeled telomeric duplex [TTAGGG]₄ with 18mer bp located adjacent to telomeric duplex is immobilized. Cy3-TERRA, Cy3-TERRA-18 or Cy3-ss18(RNA) is applied to the telomeric DNA. TERRA RNA consists of [UUAGGG]₄, and 18 is a preceding RNA sequence complementary to the 18bp located adjacent to the telomeric duplex. The Cy3-G4, Cy3-G4-18 and ss18 DNA are the DNA counterparts to Cy3-TERRA, Cy3-TERRA-18 and ss18 RNA, respectively applied to the immobilized DNA. (B) Colocalization between the Cy3 and Cy5 serves as a readout to quantify binding of TERRA to the telomeric duplex. (C) Comparison of the annealing of TERRA-18, TERRA, ss18(RNA) or ssU40 to a telomeric duplex with 18mer base. (D) Comparison of the annealing of TERRA-18, TERRA and ssRNA to the DNA counterpart G4-18-DNA, G4-DNA and ssDNA, respectively. (E) DNA construct: Cy5 telomeric duplex [TTAGGG]₄ with 15q (18) bp located adjacent to telomeric duplex. Cy3-TERRA- 15q (18) consisting of [UUAGGG]₄ and a precedint 15q(18) sequence complementary to the 15q 18bp located adjacent to telomeric duplex is applied to the immobilized DNA. (F) The fraction of colocalization of TERRA-15q and telomeric duplex with 15q (18) base. (G) Histogram for the binding TERRA-15q (18) at 2, 4,10 and 30 nM concentrations to telomeric duplex with 15q (18) basepairs.

Figure 2.

TERRA without sub-telomeric sequence exhibits dynamic states that are stabilized by TRF2. (A–D) smFRET assay shows high (0.9) stable FRET when Cy3-TERRA-18 is applied to Cy5-telomeric DNA. (E, F, H) Cy3-TERRA application to the DNA yields three FRET histogram peaks. (G, L) The individual smFRET traces undergo FRET transition and dynamics. (I, J) FRET peak distribution In the presence of TRF2. (K, L) The individual smFRET traces with the addition of TRF2.

Figure 3.

TERRA annealing results in RNA-DNA hybrid formation without displacing the G-rich strand. (A, B) Cy3-TERRA-18 is applied to Cy5 telomeric DNA. SmFRET histograms show loss of high FRET after TERRA is digested by RNase H. (C, D) Unlabeled TERRA-18 (1 uM) applied to Cy3–Cy5 labeled telomeric DNA. Sm (FRET) histogram and traces show stable high FRET before and after unlabeled TERRA-18 is applied. (E) The molecule count of the Cy3-Cy5 duplex remains the same before and after the application of excess unlabeled 18-TERRA. (F) The G-rich strand is labeled with Cy3 and Cy5 (Cy5- [TTAGGG]₄ -Cy3), and excess unlabeled TERRA is applied. Schematic shows prediction that low FRET remains if G-quadruplex does not form in DNA, and transition to a high FRET if G-quadruplex structure forms. (G) Upon applying excess unlabeled TERRA-18 and incubating with 100 or 150 mM KCl the FRET remains in the low state. (H) The same measurement as in panel C performed in ensemble fluorescence. Green and red lines represent fluorescence signals obtained from Cy3 and Cy5 over time.

Figure 4.

Structural variations of the DNA-TERRA complex revealed by combining coarse-grained and all-atom simulations. (A–C) Three representative structures of the system based on clustering the molecular simulation results. Strand-strand interactions are demonstrated by a schematic, highlighting R-loop and triplex formation. (D–F) A close-up view of the R-loop region.

Figure 5.

Structural variations of the DNA-TERRA complex revealed by combining coarse-grained and all-atom simulations. (A) FRET construct to monitor R- loop (mid-FRET) and G4 (high FRET) formation. (B) FRET level before transcription, in 20 minutes and 30 minutes of transcription and after RNase H treatment. (C) Real-time single molecule traces for DNA-only, low to mid FRET transition for R-loop formation, low to mid to high stepwise FRET transition from DNA to R-loop to G4 and mid to low FRET transition upon RNase H digestion of the R-loop.

Figure 6.

Telomeric DNA overhang and an 8oxoG lesion in the telomere duplex enhances TERRA binding. (A) Cy3 labeled TERRA or TERRA-18 was applied to telomeric duplex with or without various overhangs (T24, G2, G3, G4) constructs that are Cy5 labeled. Cy3-TERRA-18 was applied to the telomeric duplex with G4 overhang prebound to POT1 and telomeric duplex with an 8oxoG damage and Cy5 labeled. (B, C) Fraction of colocalization between TERRA-18 or TERRA with the different DNA constructs and G4 overhang prebound to POT1 as indicated. (D) Fraction of colocalization of TERRA-18 with telomeric duplex with or without 8oxoG damage.

Figure 7.

TRF2 promotes TERRA binding and protects TERRA R-loop from RNase H digestion. (A) Schematic of Cy3 labeled TERRA-18 applied to telomeric duplex prebound to TRF2, TRF1, or TRF2ΔB. (B) Colocalized fraction collected in varying protein conditions. (C) Experimental procedure of RNase H treatment alone (top) vs. proteinase K, followed by RNase H treatment (bottom). Proteinase K treatment is used to degrade the bound proteins. (D) The colocalization fraction was collected before and after the RNase H or proteinase K + RNase H treatment in all conditions. The error bar shows a standard deviation of the mean (n = 21).

Figure 8.

RAD51 binds TERRA with high affinity without unfolding the G-quadruplex structure and promotes its annealing to the telomere. (A) EMSA gel showing RAD51 binding to TERRA-18, G4-18 DNA, G4 -18–8oxoG, poly 40 oligos. (B, C) SM colocalization comparing annealing of TERRA-18 to the telomeric duplex with and without precomplexing with RAD51 protein. (D, E) SM colocalization comparing annealing of G4-18 DNA to the telomeric duplex with and without precomplexing with RAD51 protein. (F, G) TERRA pre-complexed to RAD51 colocalization with telomere DNA and RNase H treatment pre and post-proteinase K digestion of RAD51. The error bar shows a standard deviation of the mean (n = 21).