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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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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.
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gkr1306-F5: 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.

Mentions: In Figure 2A, we demonstrate that hTR1–17 is the smallest fragment capable of forming a stable quadruplex. Analyzing the sequence involved in formation of the P1 helix reveals that nucleotides 1–17 correspond to the region that is not involved in interactions with 25P1 (Figure 5B). To assess whether the quadruplex consisting of the first four guanine tracts is responsible for inhibition of P1 helix formation, we prepared truncations from the 5′-end of hTR that disrupt formation of the 1–17 quadruplex but leave the region involved in P1 helix formation intact. Interactions of each of these truncations with 25P1 were assessed by electrophoretic mobility shift assays under buffer conditions containing KCl or LiCl. While KCl stabilizes the quadruplex structure, previous work has demonstrated that substitution of LiCl is sufficient to destabilize the quadruplex structure (24). Therefore, all truncations were expected to form an interaction with 25P1 in the presence of LiCl but not KCl unless the inhibitory quadruplex was abolished by truncation. As is shown in Figure 5A, hTR1–43, hTR4–43 and hTR10–43 do not interact with 25P1 in the presence of KCl, but show nearly complete interaction in the presence of LiCl. This suggests that a quadruplex exists in these RNAs that prevents interaction with 25P1. In addition, these results suggest that the inhibitory quadruplex forms independent of the first two guanine tracts and that deletion of the third guanine tract (hTR14–43) results in disruption of the inhibitory quadruplex. This is evident in that hTR14–43 forms a complex with 25P1 in the presence of KCl. Substitution of LiCl promotes complete interaction with 25P1, indicating that a quadruplex involving the remaining four guanine tracts may persist to some degree. In summary, while the first 17 nt are sufficient to form a stable quadruplex, the major structure inhibiting P1 helix formation in hTR1–43 appears to be a quadruplex between nucleotides 11 and 28.Figure 5.


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC3351167&req=5

gkr1306-F5: 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.
Mentions: In Figure 2A, we demonstrate that hTR1–17 is the smallest fragment capable of forming a stable quadruplex. Analyzing the sequence involved in formation of the P1 helix reveals that nucleotides 1–17 correspond to the region that is not involved in interactions with 25P1 (Figure 5B). To assess whether the quadruplex consisting of the first four guanine tracts is responsible for inhibition of P1 helix formation, we prepared truncations from the 5′-end of hTR that disrupt formation of the 1–17 quadruplex but leave the region involved in P1 helix formation intact. Interactions of each of these truncations with 25P1 were assessed by electrophoretic mobility shift assays under buffer conditions containing KCl or LiCl. While KCl stabilizes the quadruplex structure, previous work has demonstrated that substitution of LiCl is sufficient to destabilize the quadruplex structure (24). Therefore, all truncations were expected to form an interaction with 25P1 in the presence of LiCl but not KCl unless the inhibitory quadruplex was abolished by truncation. As is shown in Figure 5A, hTR1–43, hTR4–43 and hTR10–43 do not interact with 25P1 in the presence of KCl, but show nearly complete interaction in the presence of LiCl. This suggests that a quadruplex exists in these RNAs that prevents interaction with 25P1. In addition, these results suggest that the inhibitory quadruplex forms independent of the first two guanine tracts and that deletion of the third guanine tract (hTR14–43) results in disruption of the inhibitory quadruplex. This is evident in that hTR14–43 forms a complex with 25P1 in the presence of KCl. Substitution of LiCl promotes complete interaction with 25P1, indicating that a quadruplex involving the remaining four guanine tracts may persist to some degree. In summary, while the first 17 nt are sufficient to form a stable quadruplex, the major structure inhibiting P1 helix formation in hTR1–43 appears to be a quadruplex between nucleotides 11 and 28.Figure 5.

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