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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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hTR1–17 is the minimal sequence capable of forming a G4-quadruplex and hTR1–20 is the smallest truncation that demonstrates affinity for endogenous RHAU. (A) Approximately 200 pmols of each hTR RNA truncation was separated by native TBE polyacrylamide gel electrophoresis and stained with 1 µg/ml n-methyl mesoporphyrin IX diluted in 20 mM Tris pH 7.5, 100 mM KCl and 1 mM EDTA for 15 min at room temperature. The gel was imaged on a Fluorchem Q system using the Cy3 excitation and emission filters. (B) Approximately 5 pmols of each hTR RNA truncation was separated as in (A) and the gel was stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (C) Approximately 5 pmols of each hTR RNA truncation was heated at 95°C for 5 min in 1× denaturing load dye and separated by denaturing TBE polyacrylamide gel electrophoresis and stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (D) Western blot of proteins enriched by streptavidin pull-down assays performed with biotinylated hTR truncations. 3′ biotinylated hTR truncations were incubated with HEK293T whole cell extracts for 30 min and protein/RNA complexes were pulled-down with 50 µl streptavidin agarose beads. The beads were boiled for 5 min in 1× SDS loading dye and the binding of RHAU to each RNA was assessed by performing SDS/PAGE and western blotting. As a control for binding specificity, blots were reprobed with anti-PKR antibodies and to control for non-specific interactions with the streptavidin agarose, a beads alone (no RNA) control was performed. Biotinylation efficiency for each RNA is demonstrated by a dot blot of 5 pmols of each RNA detected with streptavidin-HRP. (E) Sequences of each of the RNAs used for the streptavidin pull-down assay. G to C substitutions made in 43MUT are indicated by arrows. (F) Schematic of the hTR RNA based upon the published proposed secondary structure highlighting the P1 helix and overlapping quadruplex forming guanine tracts (26).
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gkr1306-F2: hTR1–17 is the minimal sequence capable of forming a G4-quadruplex and hTR1–20 is the smallest truncation that demonstrates affinity for endogenous RHAU. (A) Approximately 200 pmols of each hTR RNA truncation was separated by native TBE polyacrylamide gel electrophoresis and stained with 1 µg/ml n-methyl mesoporphyrin IX diluted in 20 mM Tris pH 7.5, 100 mM KCl and 1 mM EDTA for 15 min at room temperature. The gel was imaged on a Fluorchem Q system using the Cy3 excitation and emission filters. (B) Approximately 5 pmols of each hTR RNA truncation was separated as in (A) and the gel was stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (C) Approximately 5 pmols of each hTR RNA truncation was heated at 95°C for 5 min in 1× denaturing load dye and separated by denaturing TBE polyacrylamide gel electrophoresis and stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (D) Western blot of proteins enriched by streptavidin pull-down assays performed with biotinylated hTR truncations. 3′ biotinylated hTR truncations were incubated with HEK293T whole cell extracts for 30 min and protein/RNA complexes were pulled-down with 50 µl streptavidin agarose beads. The beads were boiled for 5 min in 1× SDS loading dye and the binding of RHAU to each RNA was assessed by performing SDS/PAGE and western blotting. As a control for binding specificity, blots were reprobed with anti-PKR antibodies and to control for non-specific interactions with the streptavidin agarose, a beads alone (no RNA) control was performed. Biotinylation efficiency for each RNA is demonstrated by a dot blot of 5 pmols of each RNA detected with streptavidin-HRP. (E) Sequences of each of the RNAs used for the streptavidin pull-down assay. G to C substitutions made in 43MUT are indicated by arrows. (F) Schematic of the hTR RNA based upon the published proposed secondary structure highlighting the P1 helix and overlapping quadruplex forming guanine tracts (26).

Mentions: RHAU has previously been demonstrated to exhibit specificity for both DNA and RNA G4-quadruplex structures in terms of affinity and helicase activity (7,15,19). hTR contains several guanine runs in the 5′ region that are known to form quadruplex in vitro. These guanine tracts are diagrammed in the context of the hTR RNA secondary structure in Figure 2F. Quadruplex formation in this region is reported to interfere with formation of the P1 helix (24). We hypothesized that RHAU interacts with the quadruplex forming region of hTR and recently published data (30,31) as well as our own data support this hypothesis. We analyzed the interaction between the endogenously expressed protein and RNA by RNA coimmunoprecipitation experiments. To confirm the interaction between RHAU and hTR, RHAU was immunoprecipitated from HEK293T whole-cell lysates without crosslinking. Copurified RNA was isolated, followed by detection and quantification by reverse transcription and RT-PCR. Results were expressed relative to the amplification of 10 ng of a total RNA extraction. Figure 1A confirms an ∼100-fold enrichment of hTR by RHAU immunoprecipitation when compared with the GAPDH mRNA, which is only marginally enriched (<2-fold). Immunoprecipitation controls with no antibody (beads alone) or an isotype matched antibody fail to enrich either hTR or the GAPDH mRNA. A similar experiment was performed by reverse transcription followed by 25 cycles of standard PCR to allow for visualization of the products on an agarose gel. Figure 1B demonstrates a single product produced for each primer set with specific enrichment of hTR by RHAU immunoprecipitation with no enrichment observed for any of the negative controls. These results support and confirm an interaction between endogenously expressed RHAU and hTR.Figure 1.


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)

hTR1–17 is the minimal sequence capable of forming a G4-quadruplex and hTR1–20 is the smallest truncation that demonstrates affinity for endogenous RHAU. (A) Approximately 200 pmols of each hTR RNA truncation was separated by native TBE polyacrylamide gel electrophoresis and stained with 1 µg/ml n-methyl mesoporphyrin IX diluted in 20 mM Tris pH 7.5, 100 mM KCl and 1 mM EDTA for 15 min at room temperature. The gel was imaged on a Fluorchem Q system using the Cy3 excitation and emission filters. (B) Approximately 5 pmols of each hTR RNA truncation was separated as in (A) and the gel was stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (C) Approximately 5 pmols of each hTR RNA truncation was heated at 95°C for 5 min in 1× denaturing load dye and separated by denaturing TBE polyacrylamide gel electrophoresis and stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (D) Western blot of proteins enriched by streptavidin pull-down assays performed with biotinylated hTR truncations. 3′ biotinylated hTR truncations were incubated with HEK293T whole cell extracts for 30 min and protein/RNA complexes were pulled-down with 50 µl streptavidin agarose beads. The beads were boiled for 5 min in 1× SDS loading dye and the binding of RHAU to each RNA was assessed by performing SDS/PAGE and western blotting. As a control for binding specificity, blots were reprobed with anti-PKR antibodies and to control for non-specific interactions with the streptavidin agarose, a beads alone (no RNA) control was performed. Biotinylation efficiency for each RNA is demonstrated by a dot blot of 5 pmols of each RNA detected with streptavidin-HRP. (E) Sequences of each of the RNAs used for the streptavidin pull-down assay. G to C substitutions made in 43MUT are indicated by arrows. (F) Schematic of the hTR RNA based upon the published proposed secondary structure highlighting the P1 helix and overlapping quadruplex forming guanine tracts (26).
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gkr1306-F2: hTR1–17 is the minimal sequence capable of forming a G4-quadruplex and hTR1–20 is the smallest truncation that demonstrates affinity for endogenous RHAU. (A) Approximately 200 pmols of each hTR RNA truncation was separated by native TBE polyacrylamide gel electrophoresis and stained with 1 µg/ml n-methyl mesoporphyrin IX diluted in 20 mM Tris pH 7.5, 100 mM KCl and 1 mM EDTA for 15 min at room temperature. The gel was imaged on a Fluorchem Q system using the Cy3 excitation and emission filters. (B) Approximately 5 pmols of each hTR RNA truncation was separated as in (A) and the gel was stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (C) Approximately 5 pmols of each hTR RNA truncation was heated at 95°C for 5 min in 1× denaturing load dye and separated by denaturing TBE polyacrylamide gel electrophoresis and stained with the total RNA stain SYBR Gold according to the manufacturer’s protocol. (D) Western blot of proteins enriched by streptavidin pull-down assays performed with biotinylated hTR truncations. 3′ biotinylated hTR truncations were incubated with HEK293T whole cell extracts for 30 min and protein/RNA complexes were pulled-down with 50 µl streptavidin agarose beads. The beads were boiled for 5 min in 1× SDS loading dye and the binding of RHAU to each RNA was assessed by performing SDS/PAGE and western blotting. As a control for binding specificity, blots were reprobed with anti-PKR antibodies and to control for non-specific interactions with the streptavidin agarose, a beads alone (no RNA) control was performed. Biotinylation efficiency for each RNA is demonstrated by a dot blot of 5 pmols of each RNA detected with streptavidin-HRP. (E) Sequences of each of the RNAs used for the streptavidin pull-down assay. G to C substitutions made in 43MUT are indicated by arrows. (F) Schematic of the hTR RNA based upon the published proposed secondary structure highlighting the P1 helix and overlapping quadruplex forming guanine tracts (26).
Mentions: RHAU has previously been demonstrated to exhibit specificity for both DNA and RNA G4-quadruplex structures in terms of affinity and helicase activity (7,15,19). hTR contains several guanine runs in the 5′ region that are known to form quadruplex in vitro. These guanine tracts are diagrammed in the context of the hTR RNA secondary structure in Figure 2F. Quadruplex formation in this region is reported to interfere with formation of the P1 helix (24). We hypothesized that RHAU interacts with the quadruplex forming region of hTR and recently published data (30,31) as well as our own data support this hypothesis. We analyzed the interaction between the endogenously expressed protein and RNA by RNA coimmunoprecipitation experiments. To confirm the interaction between RHAU and hTR, RHAU was immunoprecipitated from HEK293T whole-cell lysates without crosslinking. Copurified RNA was isolated, followed by detection and quantification by reverse transcription and RT-PCR. Results were expressed relative to the amplification of 10 ng of a total RNA extraction. Figure 1A confirms an ∼100-fold enrichment of hTR by RHAU immunoprecipitation when compared with the GAPDH mRNA, which is only marginally enriched (<2-fold). Immunoprecipitation controls with no antibody (beads alone) or an isotype matched antibody fail to enrich either hTR or the GAPDH mRNA. A similar experiment was performed by reverse transcription followed by 25 cycles of standard PCR to allow for visualization of the products on an agarose gel. Figure 1B demonstrates a single product produced for each primer set with specific enrichment of hTR by RHAU immunoprecipitation with no enrichment observed for any of the negative controls. These results support and confirm an interaction between endogenously expressed RHAU and hTR.Figure 1.

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