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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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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.
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gkr1306-F7: 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.

Mentions: Previous reports assume that a quadruplex in the 5′ region of hTR and the P1 helix are mutually exclusive structures. The data presented suggests that the 1–17 quadruplex is not the structure responsible for P1 helix inhibition and that an internal quadruplex, formed with guanines 11–13, 15–17, 21–23 and 26–28, represents the primary inhibitory structure. As we previously demonstrated that hTR1–17 is capable of forming quadruplex, and as the first 17 nt do not base pair in the P1 helix, we set out to determine if a quadruplex in the first 17 nt could persist in the presence of the P1 helix. hTR1–43 as well as the 5′ truncations hTR4–43, hTR10–43 and hTR14–43 were heated to 95°C in the presence of a 2-fold molar excess of 25P1 to induce the formation of the P1 helix. The free RNAs as well as the hTR-25P1 duplexes were then separated by native TBE polyacrylamide gel electrophoresis and stained with the quadruplex specific dye n-methyl mesoporphyrin IX. As is shown in Figure 7A, the free RNAs hTR1–43, hTR4–43 and hTR10–43 all stain with similar intensity (quantified in Figure 7C) and a significant reduction in staining is observed for hTR14–43. This result is expected based both upon the primary sequence of the truncations (Figure 5B) and our data which demonstrates that hTR14–43 does not form a stable inhibitory quadruplex (Figure 6). As is expected, 25P1 alone (lane 5) demonstrates no detectable staining for the quadruplex specific dye. When hTR1–43-25P1 complex is formed (lane 6), the staining intensity is nearly identical to hTR1–43 alone (lane 1). The 25P1 complex formed with hTR4–43 demonstrates significantly reduced quadruplex-specific staining when compared with hTR1–43, which is expected as only three guanine runs remain single stranded in the hTR4–43-25P1 duplex. As expected, the complex formed with hTR14–43 is undetectable by staining with n-methyl mesoporphyrin IX. Unexpectedly, the duplex formed with hTR10–43 still shows ∼70% of the staining intensity of the free RNA, suggesting the possibility that the remaining sequence adopts a quadruplex structure. The same gel was subsequently stained with toluidine blue to reveal total RNA. As was expected, toluidine blue stained the smaller truncations with reduced intensity as they were present in equimolar quantities. These data suggest that the free nucleotides 1–17 in the hTR1–43-25P1 complex form a stable quadruplex that is abolished upon deletion of the first guanine tract.Figure 7.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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gkr1306-F7: 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.
Mentions: Previous reports assume that a quadruplex in the 5′ region of hTR and the P1 helix are mutually exclusive structures. The data presented suggests that the 1–17 quadruplex is not the structure responsible for P1 helix inhibition and that an internal quadruplex, formed with guanines 11–13, 15–17, 21–23 and 26–28, represents the primary inhibitory structure. As we previously demonstrated that hTR1–17 is capable of forming quadruplex, and as the first 17 nt do not base pair in the P1 helix, we set out to determine if a quadruplex in the first 17 nt could persist in the presence of the P1 helix. hTR1–43 as well as the 5′ truncations hTR4–43, hTR10–43 and hTR14–43 were heated to 95°C in the presence of a 2-fold molar excess of 25P1 to induce the formation of the P1 helix. The free RNAs as well as the hTR-25P1 duplexes were then separated by native TBE polyacrylamide gel electrophoresis and stained with the quadruplex specific dye n-methyl mesoporphyrin IX. As is shown in Figure 7A, the free RNAs hTR1–43, hTR4–43 and hTR10–43 all stain with similar intensity (quantified in Figure 7C) and a significant reduction in staining is observed for hTR14–43. This result is expected based both upon the primary sequence of the truncations (Figure 5B) and our data which demonstrates that hTR14–43 does not form a stable inhibitory quadruplex (Figure 6). As is expected, 25P1 alone (lane 5) demonstrates no detectable staining for the quadruplex specific dye. When hTR1–43-25P1 complex is formed (lane 6), the staining intensity is nearly identical to hTR1–43 alone (lane 1). The 25P1 complex formed with hTR4–43 demonstrates significantly reduced quadruplex-specific staining when compared with hTR1–43, which is expected as only three guanine runs remain single stranded in the hTR4–43-25P1 duplex. As expected, the complex formed with hTR14–43 is undetectable by staining with n-methyl mesoporphyrin IX. Unexpectedly, the duplex formed with hTR10–43 still shows ∼70% of the staining intensity of the free RNA, suggesting the possibility that the remaining sequence adopts a quadruplex structure. The same gel was subsequently stained with toluidine blue to reveal total RNA. As was expected, toluidine blue stained the smaller truncations with reduced intensity as they were present in equimolar quantities. These data suggest that the free nucleotides 1–17 in the hTR1–43-25P1 complex form a stable quadruplex that is abolished upon deletion of the first guanine tract.Figure 7.

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