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The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary.

Booy EP, Meier M, Okun N, Novakowski SK, Xiong S, Stetefeld J, McKenna SA - Nucleic Acids Res. (2012)

Bottom Line: RNA associated with AU-rich element (RHAU) is an RNA helicase that has specificity for DNA and RNA G4-quadruplexes.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.

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).

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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 Kd was performed by a previously published method (34). Additional gel images are provided in Supplementary Figure S1.
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gkr1306-F3: 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 Kd was performed by a previously published method (34). Additional gel images are provided in Supplementary Figure S1.

Mentions: It has been well established that a region within the N-terminus of RHAU, referred to as the RSM, plays an essential role in the recognition and binding to G4-quadruplexes (19). To determine if the region corresponding to the RSM was sufficient for interacting with hTR, we performed electrophoretic mobility shift assays with an N-terminal truncation of RHAU consisting of amino acids 53–105 (RHAU53–105). Interactions were probed with the first 43 nt of hTR (hTR1–43) as well as a version of hTR1–43 that contained four G to C substitutions to disrupt quadruplex formation (hTR43MUT). RHAU53–105 was selected based upon predictions indicating minimal impact on secondary structure elements as well as elimination of an N-terminal glycine-rich region which may impede future structural analysis. hTR was maintained at a constant concentration of 200 nM in a 25 -µl binding reaction with increasing concentrations of RHAU53–105. Figure 3 demonstrates an interaction between hTR1–43 with ∼80% of the RNA in complex when RHAU53–105 is present at a 4-fold molar excess. Similar interactions with RHAU53–105 were obtained with the truncations hTR1–29, hTR1–24, hTR1–20, hTR1–18 and hTR1–17 (data not shown). This is in contrast to hTR43MUT in which no significant binding takes place over the range of RHAU53–105 concentrations. Quantification data (Figure 3B) is based upon densitometry analysis of three independent experiments (Supplementary Figure 1). Using a previously published method, the data reveal a dissociation constant of 310 nM for the hTR1–43-RHAU53–105 interaction (34). This suggests the interaction between RHAU and hTR is both quadruplex dependent and mediated through the RSM.Figure 3.


The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary.

Booy EP, Meier M, Okun N, Novakowski SK, Xiong S, Stetefeld J, McKenna SA - Nucleic Acids Res. (2012)

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 Kd was performed by a previously published method (34). Additional gel images are provided in Supplementary Figure S1.
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Related In: Results  -  Collection

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gkr1306-F3: 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 Kd was performed by a previously published method (34). Additional gel images are provided in Supplementary Figure S1.
Mentions: It has been well established that a region within the N-terminus of RHAU, referred to as the RSM, plays an essential role in the recognition and binding to G4-quadruplexes (19). To determine if the region corresponding to the RSM was sufficient for interacting with hTR, we performed electrophoretic mobility shift assays with an N-terminal truncation of RHAU consisting of amino acids 53–105 (RHAU53–105). Interactions were probed with the first 43 nt of hTR (hTR1–43) as well as a version of hTR1–43 that contained four G to C substitutions to disrupt quadruplex formation (hTR43MUT). RHAU53–105 was selected based upon predictions indicating minimal impact on secondary structure elements as well as elimination of an N-terminal glycine-rich region which may impede future structural analysis. hTR was maintained at a constant concentration of 200 nM in a 25 -µl binding reaction with increasing concentrations of RHAU53–105. Figure 3 demonstrates an interaction between hTR1–43 with ∼80% of the RNA in complex when RHAU53–105 is present at a 4-fold molar excess. Similar interactions with RHAU53–105 were obtained with the truncations hTR1–29, hTR1–24, hTR1–20, hTR1–18 and hTR1–17 (data not shown). This is in contrast to hTR43MUT in which no significant binding takes place over the range of RHAU53–105 concentrations. Quantification data (Figure 3B) is based upon densitometry analysis of three independent experiments (Supplementary Figure 1). Using a previously published method, the data reveal a dissociation constant of 310 nM for the hTR1–43-RHAU53–105 interaction (34). This suggests the interaction between RHAU and hTR is both quadruplex dependent and mediated through the RSM.Figure 3.

Bottom Line: RNA associated with AU-rich element (RHAU) is an RNA helicase that has specificity for DNA and RNA G4-quadruplexes.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.

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).

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