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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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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.
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gkr1306-F4: 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.

Mentions: Quadruplex formation within the 5′ region of hTR (Figure 2F) was previously hypothesized to inhibit the base pairing necessary to form the upstream P1 helix (24). The P1 helix of hTR is a critical structural element for establishing the template boundary of reverse transcription (27). To study the function of RHAU, we purified the full-length protein from HEK293T cells (Supplementary Figure S2). Preliminary experiments demonstrated that the purified protein was capable of unwinding a synthetic intermolecular quadruplex but did not show a clear impact on hTR (Supplementary Figure S3). Studying the impact of RHAU on an intramolecular quadruplex is confounded by the likelihood that, without a method of structure stabilization, the RNA quickly refolds into a quadruplex upon dissociation of the enzyme. To circumvent this obstacle, we used a previously published method using a complementary 25 nt internal fragment of hTR, referred to as 25P1, to study the potential role for RHAU in resolution of the 5′ quadruplex to promote the formation of the P1 helix (24). 25P1 and hTR1–43 were incubated either alone or together in the presence and absence of RHAU and ATP to determine if the helicase activity of RHAU could promote hTR unwinding and annealing of 25P1 (Figure 4A and B). As is clearly shown by the electrophoretic mobility shift assay, 25P1 and hTR1–43 form a detectable level of complex when combined alone. Inclusion of RHAU does not enhance duplex formation; however, inclusion of RHAU in the presence of ATP results in a nearly 4-fold increase in formation of the P1 helix. To confirm the dependence of this observation on ATP hydrolysis, the experiment was repeated using a non-hydrolysable ATP analog AMP-PNP. Under these conditions there was no promotion of the P1 helix structure (Figure 4B). Time-course analysis of the helicase activity reveals a maximal conversion of the quadruplex RNA to the P1 duplex following 45 min incubation (Figure 4D and E). These data strongly suggest that the ATP-dependent helicase activity of RHAU renders quadruplex-prone regions of hTR accessible for base pairing with the 25P1 RNA.Figure 4.


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)

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.
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gkr1306-F4: 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.
Mentions: Quadruplex formation within the 5′ region of hTR (Figure 2F) was previously hypothesized to inhibit the base pairing necessary to form the upstream P1 helix (24). The P1 helix of hTR is a critical structural element for establishing the template boundary of reverse transcription (27). To study the function of RHAU, we purified the full-length protein from HEK293T cells (Supplementary Figure S2). Preliminary experiments demonstrated that the purified protein was capable of unwinding a synthetic intermolecular quadruplex but did not show a clear impact on hTR (Supplementary Figure S3). Studying the impact of RHAU on an intramolecular quadruplex is confounded by the likelihood that, without a method of structure stabilization, the RNA quickly refolds into a quadruplex upon dissociation of the enzyme. To circumvent this obstacle, we used a previously published method using a complementary 25 nt internal fragment of hTR, referred to as 25P1, to study the potential role for RHAU in resolution of the 5′ quadruplex to promote the formation of the P1 helix (24). 25P1 and hTR1–43 were incubated either alone or together in the presence and absence of RHAU and ATP to determine if the helicase activity of RHAU could promote hTR unwinding and annealing of 25P1 (Figure 4A and B). As is clearly shown by the electrophoretic mobility shift assay, 25P1 and hTR1–43 form a detectable level of complex when combined alone. Inclusion of RHAU does not enhance duplex formation; however, inclusion of RHAU in the presence of ATP results in a nearly 4-fold increase in formation of the P1 helix. To confirm the dependence of this observation on ATP hydrolysis, the experiment was repeated using a non-hydrolysable ATP analog AMP-PNP. Under these conditions there was no promotion of the P1 helix structure (Figure 4B). Time-course analysis of the helicase activity reveals a maximal conversion of the quadruplex RNA to the P1 duplex following 45 min incubation (Figure 4D and E). These data strongly suggest that the ATP-dependent helicase activity of RHAU renders quadruplex-prone regions of hTR accessible for base pairing with the 25P1 RNA.Figure 4.

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).

Show MeSH
Related in: MedlinePlus