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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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RHAU interacts with hTR. (A) RT-PCR quantification of RNA immunoprecipitations. RHAU was immunoprecipitated from 500 µg of HEK293T whole cell lysate and the coprecipitated RNA was isolated and purified using the Qiagen RNEasy MinElute kit. As a negative control, an immunoprecipitation was performed with no antibody (beads alone) as well as an isotype matched antibody. Reverse transcription and RT-PCR were performed with primers specific for hTR or, as a control for specificity, GAPDH. Coprecipitated RNA of 2 µL (10%) was analyzed and measured in triplicate. RHAU immunoprecipitation resulted in a ∼100-fold enrichment of the hTR RNA, whereas the GAPDH RNA was enriched ∼2-fold. Fold enrichment was calculated by the comparative CT method relative to 10 ng of a total RNA extraction. Data represents the mean + standard error. (B) A similar experiment as in (A); with the exception that standard PCR was carried out following reverse transcription for 25 cycles and products were analyzed by agarose gel electrophoresis. Total RNA of 100 ng was used as a positive control.
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gkr1306-F1: RHAU interacts with hTR. (A) RT-PCR quantification of RNA immunoprecipitations. RHAU was immunoprecipitated from 500 µg of HEK293T whole cell lysate and the coprecipitated RNA was isolated and purified using the Qiagen RNEasy MinElute kit. As a negative control, an immunoprecipitation was performed with no antibody (beads alone) as well as an isotype matched antibody. Reverse transcription and RT-PCR were performed with primers specific for hTR or, as a control for specificity, GAPDH. Coprecipitated RNA of 2 µL (10%) was analyzed and measured in triplicate. RHAU immunoprecipitation resulted in a ∼100-fold enrichment of the hTR RNA, whereas the GAPDH RNA was enriched ∼2-fold. Fold enrichment was calculated by the comparative CT method relative to 10 ng of a total RNA extraction. Data represents the mean + standard error. (B) A similar experiment as in (A); with the exception that standard PCR was carried out following reverse transcription for 25 cycles and products were analyzed by agarose gel electrophoresis. Total RNA of 100 ng was used as a positive control.

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)

RHAU interacts with hTR. (A) RT-PCR quantification of RNA immunoprecipitations. RHAU was immunoprecipitated from 500 µg of HEK293T whole cell lysate and the coprecipitated RNA was isolated and purified using the Qiagen RNEasy MinElute kit. As a negative control, an immunoprecipitation was performed with no antibody (beads alone) as well as an isotype matched antibody. Reverse transcription and RT-PCR were performed with primers specific for hTR or, as a control for specificity, GAPDH. Coprecipitated RNA of 2 µL (10%) was analyzed and measured in triplicate. RHAU immunoprecipitation resulted in a ∼100-fold enrichment of the hTR RNA, whereas the GAPDH RNA was enriched ∼2-fold. Fold enrichment was calculated by the comparative CT method relative to 10 ng of a total RNA extraction. Data represents the mean + standard error. (B) A similar experiment as in (A); with the exception that standard PCR was carried out following reverse transcription for 25 cycles and products were analyzed by agarose gel electrophoresis. Total RNA of 100 ng was used as a positive control.
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gkr1306-F1: RHAU interacts with hTR. (A) RT-PCR quantification of RNA immunoprecipitations. RHAU was immunoprecipitated from 500 µg of HEK293T whole cell lysate and the coprecipitated RNA was isolated and purified using the Qiagen RNEasy MinElute kit. As a negative control, an immunoprecipitation was performed with no antibody (beads alone) as well as an isotype matched antibody. Reverse transcription and RT-PCR were performed with primers specific for hTR or, as a control for specificity, GAPDH. Coprecipitated RNA of 2 µL (10%) was analyzed and measured in triplicate. RHAU immunoprecipitation resulted in a ∼100-fold enrichment of the hTR RNA, whereas the GAPDH RNA was enriched ∼2-fold. Fold enrichment was calculated by the comparative CT method relative to 10 ng of a total RNA extraction. Data represents the mean + standard error. (B) A similar experiment as in (A); with the exception that standard PCR was carried out following reverse transcription for 25 cycles and products were analyzed by agarose gel electrophoresis. Total RNA of 100 ng was used as a positive control.
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).

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