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Human RNase H1 is associated with protein P32 and is involved in mitochondrial pre-rRNA processing.

Wu H, Sun H, Liang X, Lima WF, Crooke ST - PLoS ONE (2013)

Bottom Line: P32 enhanced the cleavage activity of RNase H1 by reducing the affinity of the enzyme for the heteroduplex substrate and enhancing turnover, but had no effect on the cleavage pattern.RNase H1 and P32 were partially co-localized in mitochondria and reduction of P32 or RNase H1 levels resulted in accumulation of mitochondrial pre ribosomal RNA [12S/16S] in HeLa cells.Further, the results identify P32 as a member of the 'RNase H1 degradosome' and the key P32 enhances the enzymatic efficiency of human RNase H1.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, Isis Pharmaceuticals, Inc., Carlsbad, California, United States of America.

ABSTRACT
Mammalian RNase H1 has been implicated in mitochondrial DNA replication and RNA processing and is required for embryonic development. We identified the mitochondrial protein P32 that binds specifically to human RNase H1, but not human RNase H2. P32 binds human RNase H1 via the hybrid-binding domain of the enzyme at an approximately 1∶1 ratio. P32 enhanced the cleavage activity of RNase H1 by reducing the affinity of the enzyme for the heteroduplex substrate and enhancing turnover, but had no effect on the cleavage pattern. RNase H1 and P32 were partially co-localized in mitochondria and reduction of P32 or RNase H1 levels resulted in accumulation of mitochondrial pre ribosomal RNA [12S/16S] in HeLa cells. P32 also co-immunoprecipitated with MRPP1, a mitochondrial RNase P protein required for mitochondrial pre-rRNA processing. The P32-RNase H1 complex was shown to physically interact with mitochondrial DNA and pre-rRNA. These results expand the potential roles for RNase H1 to include assuring proper transcription and processing of guanosine-cytosine rich pre-ribosomal RNA in mitochondria. Further, the results identify P32 as a member of the 'RNase H1 degradosome' and the key P32 enhances the enzymatic efficiency of human RNase H1.

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P32 appears to interact with the N-terminal duplex binding domain of RNase H1.(A) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. (B) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). (C) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. Km, Vmax, and kcat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.
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pone-0071006-g003: P32 appears to interact with the N-terminal duplex binding domain of RNase H1.(A) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. (B) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). (C) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. Km, Vmax, and kcat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

Mentions: To better characterize the interaction between human RNase H1 and P32 and to further understand which regions of the enzyme interact with P32, we expressed and purified two his-tagged RNase H1 deletion mutants (Figure 3A). RNase H1 is comprised of three functional domains: the N-terminal hybrid binding domain (aa1-72), spacer domain (aa73-135), and the C-terminal catalytic domain (aa136-286) (Figure 3A, left panel). The mutant DL1 deletes the hybrid binding domain and DL2 lacks both the hybrid binding domain and the spacer region leaving the catalytic domain only, which is similar to E. coli RNase H1. Both proteins have been previously shown to exhibit strong RNase H cleavage activity [17]. Neither the DL1 nor DL2 deletion mutant appeared to bind P32 in NaCl concentrations ranging from 150-450 mM (Figure 3B). These results suggest that P32 interacts with the hybrid binding domain of human RNase H1. Consistent with these results, P32 appeared to have little or no effect on the cleavage kinetics of the DL1 mutant for the RNA/DNA substrate (Figure 3C). The slight increase in the Km, Vmax and kcat observed for DL1 in the presence of P32 was insignificant compared to the effect of P32 on the cleavage kinetics of the full length RNase H1 (Figure 2E). Together, these results support the observation that P32 interacts with the N-terminal hybrid binding domain of human RNase.


Human RNase H1 is associated with protein P32 and is involved in mitochondrial pre-rRNA processing.

Wu H, Sun H, Liang X, Lima WF, Crooke ST - PLoS ONE (2013)

P32 appears to interact with the N-terminal duplex binding domain of RNase H1.(A) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. (B) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). (C) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. Km, Vmax, and kcat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.
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pone-0071006-g003: P32 appears to interact with the N-terminal duplex binding domain of RNase H1.(A) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. (B) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). (C) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. Km, Vmax, and kcat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.
Mentions: To better characterize the interaction between human RNase H1 and P32 and to further understand which regions of the enzyme interact with P32, we expressed and purified two his-tagged RNase H1 deletion mutants (Figure 3A). RNase H1 is comprised of three functional domains: the N-terminal hybrid binding domain (aa1-72), spacer domain (aa73-135), and the C-terminal catalytic domain (aa136-286) (Figure 3A, left panel). The mutant DL1 deletes the hybrid binding domain and DL2 lacks both the hybrid binding domain and the spacer region leaving the catalytic domain only, which is similar to E. coli RNase H1. Both proteins have been previously shown to exhibit strong RNase H cleavage activity [17]. Neither the DL1 nor DL2 deletion mutant appeared to bind P32 in NaCl concentrations ranging from 150-450 mM (Figure 3B). These results suggest that P32 interacts with the hybrid binding domain of human RNase H1. Consistent with these results, P32 appeared to have little or no effect on the cleavage kinetics of the DL1 mutant for the RNA/DNA substrate (Figure 3C). The slight increase in the Km, Vmax and kcat observed for DL1 in the presence of P32 was insignificant compared to the effect of P32 on the cleavage kinetics of the full length RNase H1 (Figure 2E). Together, these results support the observation that P32 interacts with the N-terminal hybrid binding domain of human RNase.

Bottom Line: P32 enhanced the cleavage activity of RNase H1 by reducing the affinity of the enzyme for the heteroduplex substrate and enhancing turnover, but had no effect on the cleavage pattern.RNase H1 and P32 were partially co-localized in mitochondria and reduction of P32 or RNase H1 levels resulted in accumulation of mitochondrial pre ribosomal RNA [12S/16S] in HeLa cells.Further, the results identify P32 as a member of the 'RNase H1 degradosome' and the key P32 enhances the enzymatic efficiency of human RNase H1.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, Isis Pharmaceuticals, Inc., Carlsbad, California, United States of America.

ABSTRACT
Mammalian RNase H1 has been implicated in mitochondrial DNA replication and RNA processing and is required for embryonic development. We identified the mitochondrial protein P32 that binds specifically to human RNase H1, but not human RNase H2. P32 binds human RNase H1 via the hybrid-binding domain of the enzyme at an approximately 1∶1 ratio. P32 enhanced the cleavage activity of RNase H1 by reducing the affinity of the enzyme for the heteroduplex substrate and enhancing turnover, but had no effect on the cleavage pattern. RNase H1 and P32 were partially co-localized in mitochondria and reduction of P32 or RNase H1 levels resulted in accumulation of mitochondrial pre ribosomal RNA [12S/16S] in HeLa cells. P32 also co-immunoprecipitated with MRPP1, a mitochondrial RNase P protein required for mitochondrial pre-rRNA processing. The P32-RNase H1 complex was shown to physically interact with mitochondrial DNA and pre-rRNA. These results expand the potential roles for RNase H1 to include assuring proper transcription and processing of guanosine-cytosine rich pre-ribosomal RNA in mitochondria. Further, the results identify P32 as a member of the 'RNase H1 degradosome' and the key P32 enhances the enzymatic efficiency of human RNase H1.

Show MeSH
Related in: MedlinePlus