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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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Co-localization of P32 and RNase H1.(A) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse (green) and TRITC conjugated anti-rabbit secondary antibodies (red). Nuclei were stained with DAP1 (Blue) and Mitochondria were stained with mitotracker (white). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. (B) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.
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pone-0071006-g004: Co-localization of P32 and RNase H1.(A) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse (green) and TRITC conjugated anti-rabbit secondary antibodies (red). Nuclei were stained with DAP1 (Blue) and Mitochondria were stained with mitotracker (white). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. (B) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

Mentions: It is known that human RNase H1 is present in both the cytoplasm and nucleus [40], and P32 is predominantly located in mitochondria but a fraction of this protein can also be detected in other subcellular locations including the nucleus [47]–[51]. We determined the cellular localization of the two proteins using immunofluorescent staining for endogenous P32 and RNase H1 in HeLa cells (Figure 4A). As expected, P32 is localized predominantly in the cytoplasm (Figure 4A upper panel) in a pattern consistent with a mitochondrial location. However, a small proportion of the P32 signal was also observed in the nucleus. The mitochondrial and nuclear locations of P32 were further confirmed by isolating nuclei and mitochondria and evaluating the levels of the protein via western analysis (Figure 4B). As expected, RNase H1 localized in both the nucleus and the cytoplasm and RNase H1 co-localized with P32 in mitochondria as determined by immunoflurescence staining (Figure 4A). To further confirm this co-localization, HeLa cells were infected with an adenovirus expressing full length RNase H1. The P32 and the over-expressed RNase H1 clearly co-localized in similar regions of cytoplasm, as determined with higher resolution imaging (Figure 4A, lower panel). The presence of a small amount of P32 in the nucleus suggests that P32 and RNase H1 may also co-localize in this compartment. However, since the highly enriched RNase H1 is evenly distributed in the nucleus, it is difficult to evaluate the subnuclear localization of overexpressed RNase H1.


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)

Co-localization of P32 and RNase H1.(A) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse (green) and TRITC conjugated anti-rabbit secondary antibodies (red). Nuclei were stained with DAP1 (Blue) and Mitochondria were stained with mitotracker (white). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. (B) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750045&req=5

pone-0071006-g004: Co-localization of P32 and RNase H1.(A) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse (green) and TRITC conjugated anti-rabbit secondary antibodies (red). Nuclei were stained with DAP1 (Blue) and Mitochondria were stained with mitotracker (white). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. (B) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.
Mentions: It is known that human RNase H1 is present in both the cytoplasm and nucleus [40], and P32 is predominantly located in mitochondria but a fraction of this protein can also be detected in other subcellular locations including the nucleus [47]–[51]. We determined the cellular localization of the two proteins using immunofluorescent staining for endogenous P32 and RNase H1 in HeLa cells (Figure 4A). As expected, P32 is localized predominantly in the cytoplasm (Figure 4A upper panel) in a pattern consistent with a mitochondrial location. However, a small proportion of the P32 signal was also observed in the nucleus. The mitochondrial and nuclear locations of P32 were further confirmed by isolating nuclei and mitochondria and evaluating the levels of the protein via western analysis (Figure 4B). As expected, RNase H1 localized in both the nucleus and the cytoplasm and RNase H1 co-localized with P32 in mitochondria as determined by immunoflurescence staining (Figure 4A). To further confirm this co-localization, HeLa cells were infected with an adenovirus expressing full length RNase H1. The P32 and the over-expressed RNase H1 clearly co-localized in similar regions of cytoplasm, as determined with higher resolution imaging (Figure 4A, lower panel). The presence of a small amount of P32 in the nucleus suggests that P32 and RNase H1 may also co-localize in this compartment. However, since the highly enriched RNase H1 is evenly distributed in the nucleus, it is difficult to evaluate the subnuclear localization of overexpressed RNase H1.

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