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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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Related in: MedlinePlus

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.
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gkr1306-F6: 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.

Mentions: To assess whether RHAU is capable of unwinding the internal inhibitory quadruplex present in the hTR 5′ truncations, we assessed RHAU helicase activity on each of the RNAs in the presence of the complementary strand 25P1. hTR1–43 demonstrated a similar enhancement of the interaction with 25P1 in the presence of RHAU and ATP (Figure 6). RHAU had a similar impact on hTR4–43, but demonstrated an enhanced activity. Activity appeared further enhanced when RHAU was incubated with hTR10–43 with complete conversion of the RNA to the double-stranded P1 helix. While the majority of hTR14–43 formed a duplex with 25P1 independent of RHAU, inclusion of the helicase enhanced the interaction and resulted in complete conversion of the free RNA to the 25P1 duplex. These data demonstrate that RHAU is capable of unwinding quadruplexes within all of the hTR 5′ truncations to promote an interaction with 25P1.Figure 6.


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

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

gkr1306-F6: 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.
Mentions: To assess whether RHAU is capable of unwinding the internal inhibitory quadruplex present in the hTR 5′ truncations, we assessed RHAU helicase activity on each of the RNAs in the presence of the complementary strand 25P1. hTR1–43 demonstrated a similar enhancement of the interaction with 25P1 in the presence of RHAU and ATP (Figure 6). RHAU had a similar impact on hTR4–43, but demonstrated an enhanced activity. Activity appeared further enhanced when RHAU was incubated with hTR10–43 with complete conversion of the RNA to the double-stranded P1 helix. While the majority of hTR14–43 formed a duplex with 25P1 independent of RHAU, inclusion of the helicase enhanced the interaction and resulted in complete conversion of the free RNA to the 25P1 duplex. These data demonstrate that RHAU is capable of unwinding quadruplexes within all of the hTR 5′ truncations to promote an interaction with 25P1.Figure 6.

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