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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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Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA.HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. (A) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. (B) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. (C) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNAVal-16 S rRNA junction (pre-16 S) or to the tRNAGly-ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.
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pone-0071006-g005: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA.HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. (A) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. (B) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. (C) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNAVal-16 S rRNA junction (pre-16 S) or to the tRNAGly-ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

Mentions: To investigate the potential roles of the RNase H1-P32 complex in mitochondrial RNA expression, we tested three sets of different siRNAs which specifically targeted either RNase H1 or P32. A greater than 90% reduction of RNase H1 or P32 mRNA was observed 24 hours after siRNA treatment (Figure 5A). The protein levels of RNase H1 and P32 were reduced by over 90% and 70%, respectively, after 48 hours of treatment (Figure 5B). Reduction of either RNase H1 or P32 mRNAs or proteins appeared to have no effect on the level of the other mRNAs or proteins evaluated (data not shown). In cells depleted of RNase H1, a significant increase in the 12 S/16 S mitochondrial rRNA precursor was observed, as demonstrated by northern hybridization using either a 12 S rRNA probe (Figure 5C, upper panel) or a 16 S rRNA probe (Figure 5C, middle panel). Reduction of RNase H1 increased the precursor level by more than 60% (Figure 5C, lower panel). Interestingly, an even greater increase (>300%) of the rRNA precursor was observed in P32 depleted cells, suggesting that reducing RNase H1 or P32 does not affect mtDNA transcription, however, subsequent RNA processing was impaired. This view is further supported by qRT-PCR results showing that reduction of RNase H1 or P32 caused accumulation of pre-16 S rRNA, whereas the level of the pre-ND3 region, which is part of the polycistronic transcript from the heavy strand, was not significantly affected (Figure 5D).


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)

Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA.HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. (A) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. (B) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. (C) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNAVal-16 S rRNA junction (pre-16 S) or to the tRNAGly-ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.
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Related In: Results  -  Collection

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pone-0071006-g005: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA.HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. (A) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. (B) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. (C) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNAVal-16 S rRNA junction (pre-16 S) or to the tRNAGly-ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.
Mentions: To investigate the potential roles of the RNase H1-P32 complex in mitochondrial RNA expression, we tested three sets of different siRNAs which specifically targeted either RNase H1 or P32. A greater than 90% reduction of RNase H1 or P32 mRNA was observed 24 hours after siRNA treatment (Figure 5A). The protein levels of RNase H1 and P32 were reduced by over 90% and 70%, respectively, after 48 hours of treatment (Figure 5B). Reduction of either RNase H1 or P32 mRNAs or proteins appeared to have no effect on the level of the other mRNAs or proteins evaluated (data not shown). In cells depleted of RNase H1, a significant increase in the 12 S/16 S mitochondrial rRNA precursor was observed, as demonstrated by northern hybridization using either a 12 S rRNA probe (Figure 5C, upper panel) or a 16 S rRNA probe (Figure 5C, middle panel). Reduction of RNase H1 increased the precursor level by more than 60% (Figure 5C, lower panel). Interestingly, an even greater increase (>300%) of the rRNA precursor was observed in P32 depleted cells, suggesting that reducing RNase H1 or P32 does not affect mtDNA transcription, however, subsequent RNA processing was impaired. This view is further supported by qRT-PCR results showing that reduction of RNase H1 or P32 caused accumulation of pre-16 S rRNA, whereas the level of the pre-ND3 region, which is part of the polycistronic transcript from the heavy strand, was not significantly affected (Figure 5D).

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