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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

The RHAU RSM domain interacts with the terminal quadruplex of the hTR1–43-25P1 complex. hTR1–43 and successive 5′ truncations were heated to 95°C and allowed to cool either alone or in the presence of a 2-fold molar excess of 25P1. This process converted the majority of the hTR RNA into a complex with 25P1 (hTR-25P1, upper band in lanes 3, 7, 11 and 15). RHAU53–105 was added at a 3-fold molar excess and binding reactions were incubated for 15 min at room temperature. RNAs and RNA–protein complexes were separated by native TBE polyacrylamide gel electrophoresis and stained with SYBR Gold. RHAU53–105 demonstrated an interaction with all of the hTR truncations (lanes 2, 6, 10 and 14); however, significantly decreased affinity was observed for hTR14–43 (lane 14). RHAU53–105 demonstrated an interaction with the hTR1–43-25P1 complex (lane 4, upper band). This interaction is diminished in the case of the hTR4–43-25P1 (lane 8, upper band), and abolished in the case of both hTR10–43-25P1 (lane 12) and hTR14–43-25P1 (lane 16). Faint bands not identified by arrows represent residual alternative conformations of the RNA species.
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gkr1306-F8: The RHAU RSM domain interacts with the terminal quadruplex of the hTR1–43-25P1 complex. hTR1–43 and successive 5′ truncations were heated to 95°C and allowed to cool either alone or in the presence of a 2-fold molar excess of 25P1. This process converted the majority of the hTR RNA into a complex with 25P1 (hTR-25P1, upper band in lanes 3, 7, 11 and 15). RHAU53–105 was added at a 3-fold molar excess and binding reactions were incubated for 15 min at room temperature. RNAs and RNA–protein complexes were separated by native TBE polyacrylamide gel electrophoresis and stained with SYBR Gold. RHAU53–105 demonstrated an interaction with all of the hTR truncations (lanes 2, 6, 10 and 14); however, significantly decreased affinity was observed for hTR14–43 (lane 14). RHAU53–105 demonstrated an interaction with the hTR1–43-25P1 complex (lane 4, upper band). This interaction is diminished in the case of the hTR4–43-25P1 (lane 8, upper band), and abolished in the case of both hTR10–43-25P1 (lane 12) and hTR14–43-25P1 (lane 16). Faint bands not identified by arrows represent residual alternative conformations of the RNA species.

Mentions: After confirming the presence of a quadruplex structure following P1 helix formation in hTR1–43 (Figure 7), we sought to determine whether this quadruplex retained affinity for RHAU. Electrophoretic mobility shift assays were performed with each of the hTR 5′ truncations alone or in complex with the complementary 25P1 RNA. As is clearly shown by the electrophoretic mobility shifts in Figure 8 (lanes 2, 6, 10 and 14), each of the free RNAs exhibit affinity for RHAU53–105; however hTR14–43 exhibited substantially reduced binding. Interestingly, P1 helix assembled with hTR1–43, forms a complex with RHAU53–105 (upper band, lane 4) as can be seen by the complete shift of the hTR-25P1 band seen in lane 3 to a higher molecular weight complex (lane 4). This indicates that the persisting quadruplex retains the ability to interact with RHAU. This interaction was disrupted upon truncation of the first 3 nt as hTR4–43 exhibited minimal interaction (lane 8). While all of the dominant hTR4–43-25P1 duplex observed in lane 7 remains in lane 8, a complex does form that appears to result from a subpopulation of hTR4–43-25P1 (second band from top, Figure 8, lane 7) that stained with the quadruplex-specific dye (Figure 7, lane 7), possibly due to incomplete base pairing with 25P1. In the case of hTR10–43 and hTR14–43, no interaction is evident for the hTR-25P1 complex with RHAU53–105 (lanes 12 and 16). These data mirror what was observed in Figure 7, in that RNAs or complexes demonstrating affinity for the quadruplex dye n-methyl mesoporphyrin IX also exhibit affinity for RHAU53–105.Figure 8.


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)

The RHAU RSM domain interacts with the terminal quadruplex of the hTR1–43-25P1 complex. hTR1–43 and successive 5′ truncations were heated to 95°C and allowed to cool either alone or in the presence of a 2-fold molar excess of 25P1. This process converted the majority of the hTR RNA into a complex with 25P1 (hTR-25P1, upper band in lanes 3, 7, 11 and 15). RHAU53–105 was added at a 3-fold molar excess and binding reactions were incubated for 15 min at room temperature. RNAs and RNA–protein complexes were separated by native TBE polyacrylamide gel electrophoresis and stained with SYBR Gold. RHAU53–105 demonstrated an interaction with all of the hTR truncations (lanes 2, 6, 10 and 14); however, significantly decreased affinity was observed for hTR14–43 (lane 14). RHAU53–105 demonstrated an interaction with the hTR1–43-25P1 complex (lane 4, upper band). This interaction is diminished in the case of the hTR4–43-25P1 (lane 8, upper band), and abolished in the case of both hTR10–43-25P1 (lane 12) and hTR14–43-25P1 (lane 16). Faint bands not identified by arrows represent residual alternative conformations of the RNA species.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1306-F8: The RHAU RSM domain interacts with the terminal quadruplex of the hTR1–43-25P1 complex. hTR1–43 and successive 5′ truncations were heated to 95°C and allowed to cool either alone or in the presence of a 2-fold molar excess of 25P1. This process converted the majority of the hTR RNA into a complex with 25P1 (hTR-25P1, upper band in lanes 3, 7, 11 and 15). RHAU53–105 was added at a 3-fold molar excess and binding reactions were incubated for 15 min at room temperature. RNAs and RNA–protein complexes were separated by native TBE polyacrylamide gel electrophoresis and stained with SYBR Gold. RHAU53–105 demonstrated an interaction with all of the hTR truncations (lanes 2, 6, 10 and 14); however, significantly decreased affinity was observed for hTR14–43 (lane 14). RHAU53–105 demonstrated an interaction with the hTR1–43-25P1 complex (lane 4, upper band). This interaction is diminished in the case of the hTR4–43-25P1 (lane 8, upper band), and abolished in the case of both hTR10–43-25P1 (lane 12) and hTR14–43-25P1 (lane 16). Faint bands not identified by arrows represent residual alternative conformations of the RNA species.
Mentions: After confirming the presence of a quadruplex structure following P1 helix formation in hTR1–43 (Figure 7), we sought to determine whether this quadruplex retained affinity for RHAU. Electrophoretic mobility shift assays were performed with each of the hTR 5′ truncations alone or in complex with the complementary 25P1 RNA. As is clearly shown by the electrophoretic mobility shifts in Figure 8 (lanes 2, 6, 10 and 14), each of the free RNAs exhibit affinity for RHAU53–105; however hTR14–43 exhibited substantially reduced binding. Interestingly, P1 helix assembled with hTR1–43, forms a complex with RHAU53–105 (upper band, lane 4) as can be seen by the complete shift of the hTR-25P1 band seen in lane 3 to a higher molecular weight complex (lane 4). This indicates that the persisting quadruplex retains the ability to interact with RHAU. This interaction was disrupted upon truncation of the first 3 nt as hTR4–43 exhibited minimal interaction (lane 8). While all of the dominant hTR4–43-25P1 duplex observed in lane 7 remains in lane 8, a complex does form that appears to result from a subpopulation of hTR4–43-25P1 (second band from top, Figure 8, lane 7) that stained with the quadruplex-specific dye (Figure 7, lane 7), possibly due to incomplete base pairing with 25P1. In the case of hTR10–43 and hTR14–43, no interaction is evident for the hTR-25P1 complex with RHAU53–105 (lanes 12 and 16). These data mirror what was observed in Figure 7, in that RNAs or complexes demonstrating affinity for the quadruplex dye n-methyl mesoporphyrin IX also exhibit affinity for RHAU53–105.Figure 8.

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