The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary - PubMed (original) (raw)
The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary
E P Booy et al. Nucleic Acids Res. 2012 May.
Abstract
Human telomerase RNA (hTR) contains several guanine tracts at its 5'-end that can form a G4-quadruplex structure. Previous evidence suggests that a G4-quadruplex within this region disrupts the formation of an important structure within hTR known as the P1 helix, a critical element in defining the template boundary for reverse transcription. RNA associated with AU-rich element (RHAU) is an RNA helicase that has specificity for DNA and RNA G4-quadruplexes. Two recent studies identify a specific interaction between hTR and RHAU. Herein, we confirm this interaction and identify the minimally interacting RNA fragments. We demonstrate the existence of multiple quadruplex structures within the 5' region of hTR and find that these regions parallel the minimal sequences capable of RHAU interaction. We confirm the importance of the RHAU-specific motif in the interaction with hTR and demonstrate that the helicase activity of RHAU is sufficient to unwind the quadruplex and promote an interaction with 25 internal nucleotides to form a stable P1 helix. Furthermore, we have found that a 5'-terminal quadruplex persists following P1 helix formation that retains affinity for RHAU. Finally, we have investigated the functional implications of this interaction and demonstrated a reduction in average telomere length following RHAU knockdown by small interfering RNA (siRNA).
Figures
Figure 1.
RHAU interacts with hTR. (A) RT-PCR quantification of RNA immunoprecipitations. RHAU was immunoprecipitated from 500 µg of HEK293T whole cell lysate and the coprecipitated RNA was isolated and purified using the Qiagen RNEasy MinElute kit. As a negative control, an immunoprecipitation was performed with no antibody (beads alone) as well as an isotype matched antibody. Reverse transcription and RT-PCR were performed with primers specific for hTR or, as a control for specificity, GAPDH. Coprecipitated RNA of 2 µL (10%) was analyzed and measured in triplicate. RHAU immunoprecipitation resulted in a ∼100-fold enrichment of the hTR RNA, whereas the GAPDH RNA was enriched ∼2-fold. Fold enrichment was calculated by the comparative _C_T method relative to 10 ng of a total RNA extraction. Data represents the mean + standard error. (B) A similar experiment as in (A); with the exception that standard PCR was carried out following reverse transcription for 25 cycles and products were analyzed by agarose gel electrophoresis. Total RNA of 100 ng was used as a positive control.
Figure 2.
hTR1–17 is the minimal sequence capable of forming a G4-quadruplex and hTR1–20 is the smallest truncation that demonstrates affinity for endogenous RHAU. (A) Approximately 200 pmols of each hTR RNA truncation was separated by native TBE polyacrylamide gel electrophoresis and stained with 1 µg/ml _n_-methyl mesoporphyrin IX diluted in 20 mM Tris pH 7.5, 100 mM KCl and 1 mM EDTA for 15 min at room temperature. The gel was imaged on a Fluorchem Q system using the Cy3 excitation and emission filters. (B) Approximately 5 pmols of each hTR RNA truncation was separated as in (A) and the gel was stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (C) Approximately 5 pmols of each hTR RNA truncation was heated at 95°C for 5 min in 1× denaturing load dye and separated by denaturing TBE polyacrylamide gel electrophoresis and stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (D) Western blot of proteins enriched by streptavidin pull-down assays performed with biotinylated hTR truncations. 3′ biotinylated hTR truncations were incubated with HEK293T whole cell extracts for 30 min and protein/RNA complexes were pulled-down with 50 µl streptavidin agarose beads. The beads were boiled for 5 min in 1× SDS loading dye and the binding of RHAU to each RNA was assessed by performing SDS/PAGE and western blotting. As a control for binding specificity, blots were reprobed with anti-PKR antibodies and to control for non-specific interactions with the streptavidin agarose, a beads alone (no RNA) control was performed. Biotinylation efficiency for each RNA is demonstrated by a dot blot of 5 pmols of each RNA detected with streptavidin-HRP. (E) Sequences of each of the RNAs used for the streptavidin pull-down assay. G to C substitutions made in 43MUT are indicated by arrows. (F) Schematic of the hTR RNA based upon the published proposed secondary structure highlighting the P1 helix and overlapping quadruplex forming guanine tracts (26).
Figure 3.
hTR1–43 interacts with an N-terminal RHAU truncation (RHAU53–105) containing the RSM. (A) Electrophoretic mobility shift assay demonstrating a specific interaction of RHAU53–105 with hTR1–43. 200 nM hTR RNA was incubated in a binding reaction with increasing concentrations of RHAU53–105 for 15 min at room temperature and the free RNA and RNA/protein complexes were resolved by native TBE polyacrylamide gel electrophoresis and stained with the nucleic acid dye SYBR Gold. The lower gel demonstrates a loss of interaction when G to C substitutions are introduced into the RNA sequence (hTR43MUT). (B) Quantification of the free RNA band for hTR1–43. Data represent the mean of three independent experiments ± standard deviation. Curve fitting and calculation of _K_d was performed by a previously published method (34). Additional gel images are provided in
Supplementary Figure S1
.
Figure 4.
RHAU promotes the formation of the hTR P1 helix. (A) Native TBE gel electrophoresis of the RNAs 25P1 and hTR1–43 both alone and in complex. Duplex formation was assessed with 25P1 and hTR1–43 alone and together in the presence and absence of the full length recombinant RHAU protein and either 1 mM ATP or 1 mM AMP-PNP. Approximately 200 nM of hTR1–43 was combined with 400 nM 25P1 in a 25 µl reaction ± 50 nM RHAU and 1 mM ATP/1 mM AMP-PNP for 30 min at 30°C. RNAs were separated by native electrophoresis and stained with SYBR Gold. (B) Densitometry analysis of the hTR1–43-25P1 complex band intensity relative to the RNA alone lane. Data reveals an ATP-dependent 4-fold increase in P1 helix formation in the presence of RHAU. Data represent the mean of three independent experiments ± standard deviation. Additional gel images are provided in
Supplementary Figures S4
and
S5
. (C) Schematic representing sequence details of the 25P1 RNA, hTR1–43 as well as the expected double stranded interaction product. (D) Time-course analysis of the hTR43-25P1 duplex formation in the presence of RHAU and 1 mM ATP. RHAU was added to the reaction mixture and the tubes were incubated at 30°C for the indicated time-points. RNAs were then separated by Native TBE gel electrophoresis. (E) Densitometry analysis of the hTR1–43 -25P1 complex band intensity relative to the 0 min time-point. Data represent the mean of three independent experiments ± standard deviation. Additional gel images are provided in
Supplementary Figure S6
.
Figure 5.
Deletion of the first 13 nt of the hTR RNA disrupts a quadruplex responsible for blocking P1 helix formation. (A) Electrophoretic mobility shift assays examining the formation of P1 helix in hTR RNAs containing successive truncations from the 5′-end. Each hTR truncation was incubated in the presence and absence of 25P1 in a buffer containing either 100 mM KCl or 100 mM LiCl. All RNAs with the exception of hTR14–43 failed to interact in the presence of KCl. Quadruplex disruption by LiCl resulted in nearly complete interaction of the hTR RNAs with 25P1. hTR14–43 demonstrated significant interaction in the presence of KCl that was enhanced in the presence of LiCl. The two upper bands present in the 25P1 duplex formed with hTR1–43 and hTR4–43 are likely due to alternative conformations of the single stranded RNA outside of the duplex. (B) Schematic detailing the sequence of each hTR truncation as well as the 25P1 RNA and the expected interaction site.
Figure 6.
RHAU promotes the formation of the hTR P1 helix in all hTR 5′ truncations. Binding reactions were performed for each hTR RNA truncation with a 2-fold excess of the 25P1 RNA in the presence and absence of the full length RHAU protein. RNAs were incubated at 30°C for 30 min and then resolved by native TBE polyacrylamide gel electrophoresis. RHAU strongly enhanced P1 helix formation for each of the RNAs with enhanced efficiency observed in the 5′ truncated forms.
Figure 7.
A switch between an internal and terminal quadruplex occurs upon P1 helix formation. (A) hTR1–43 and successive 5′ truncations were heated to 95°C either alone or in the presence of a 2-fold molar excess of 25P1 for 5 min and then allowed to cool to room temperature. Approximately 500 pmols of each RNA was separated by native TBE polyacrylamide gel electrophoresis and stained with the quadruplex-specific fluorescent dye _n_-methyl mesoporphyrin IX. A significant decrease in staining intensity was observed for hTR14–43 indicating a loss of quadruplex structure. The hTR1–43-25P1 complex stained with the quadruplex-specific dye; however, this staining was completely lost in the hTR14–43-25P1 complex. (B) Following fluorescent visualization of the gel it was further stained with the total RNA stain toluidine blue. (C) Densitometry quantification of the bands in (A). In the case of hTR-25P1 complexes, the dominant band, as observed in (B), was chosen for quantification. Data represents the mean of three independent experiments ± standard deviation. Additional gel images are provided in
Supplementary Figure 7
.
Figure 8.
The RHAU RSM domain interacts with the terminal quadruplex of the hTR1–43-25P1 complex. hTR1–43 and successive 5′ truncations were heated to 95°C and allowed to cool either alone or in the presence of a 2-fold molar excess of 25P1. This process converted the majority of the hTR RNA into a complex with 25P1 (hTR-25P1, upper band in lanes 3, 7, 11 and 15). RHAU53–105 was added at a 3-fold molar excess and binding reactions were incubated for 15 min at room temperature. RNAs and RNA–protein complexes were separated by native TBE polyacrylamide gel electrophoresis and stained with SYBR Gold. RHAU53–105 demonstrated an interaction with all of the hTR truncations (lanes 2, 6, 10 and 14); however, significantly decreased affinity was observed for hTR14–43 (lane 14). RHAU53–105 demonstrated an interaction with the hTR1–43-25P1 complex (lane 4, upper band). This interaction is diminished in the case of the hTR4–43-25P1 (lane 8, upper band), and abolished in the case of both hTR10–43-25P1 (lane 12) and hTR14–43-25P1 (lane 16). Faint bands not identified by arrows represent residual alternative conformations of the RNA species.
Figure 9.
RHAU knockdown by siRNA results in a reduction in average relative telomere length 7 days post-transfection that is recovered by 10 days. (A) Average relative telomere lengths were assessed by a quantitative RT-PCR-based assay using primers that generate a fixed length product and standardized to primers specific for the ALB gene (33). Data represent the average of three independent experiments measured in triplicate relative to the _C_T obtained for untransfected cells harvested at day zero. Asterisks indicates P = 0.048 at Day 7 as measured by paired one-tail _t_-test. (B) Western blot demonstrating efficiency of the RHAU siRNA knockdown. Cells harvested at each time-point were lysed in RIPA buffer and 40 µg from each time point was analyzed by SDS/PAGE followed by western blotting with antibodies specific for RHAU. Blots were reprobed with antibodies specific for β-tubulin as a loading control.
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