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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 can be co-immunoprecipitated with mitochondrial RNase P protein.(A) RT-PCR assay for the mRNA levels of P32 or MRPP1 in siRNA treated HeLa cells. The error bars represent standard deviation of three replicates. (B) Northern hybridization for mitochondrial pre-rRNA. Total RNA prepared from HeLa cells pre-treated for 24 hrs with P32 [(−)P32] or MRPP1 [(−)MRPP1] siRNAs was analyzed by northern hybridization, using probes specific to pre-rRNA, as in Figure 5.The same blot was re-probed for 7 SL RNA, which served as a loading control. (C) Immunoprecipitation using MRPP1 antibody (MRPP1-AB) or RPL5 antibody (RPL5-AB) was performed as described in materials and methods. Co-isolated proteins were separated by SDS-PAGE, and P32 protein was determined by western analysis. (−)AB, control immunoprecipitation in the absence of antibody. (D) DNase I treatment disrupted the P32-MRPP1 interaction. Immunoprecipitation was performed using MRPP1 antibody, as in panel C. After wash, the beads were incubated with either RNase A or DNase I, to elute bound materials. The eluted proteins were analyzed by western blots for the presence of P32 protein. Buffer, 1×TE buffer alone.
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pone-0071006-g007: P32 can be co-immunoprecipitated with mitochondrial RNase P protein.(A) RT-PCR assay for the mRNA levels of P32 or MRPP1 in siRNA treated HeLa cells. The error bars represent standard deviation of three replicates. (B) Northern hybridization for mitochondrial pre-rRNA. Total RNA prepared from HeLa cells pre-treated for 24 hrs with P32 [(−)P32] or MRPP1 [(−)MRPP1] siRNAs was analyzed by northern hybridization, using probes specific to pre-rRNA, as in Figure 5.The same blot was re-probed for 7 SL RNA, which served as a loading control. (C) Immunoprecipitation using MRPP1 antibody (MRPP1-AB) or RPL5 antibody (RPL5-AB) was performed as described in materials and methods. Co-isolated proteins were separated by SDS-PAGE, and P32 protein was determined by western analysis. (−)AB, control immunoprecipitation in the absence of antibody. (D) DNase I treatment disrupted the P32-MRPP1 interaction. Immunoprecipitation was performed using MRPP1 antibody, as in panel C. After wash, the beads were incubated with either RNase A or DNase I, to elute bound materials. The eluted proteins were analyzed by western blots for the presence of P32 protein. Buffer, 1×TE buffer alone.

Mentions: Since P32 itself does not contain nuclease domains and RNase H1 acts on DNA/RNA duplexes, the involvement of P32 and RNase H1 in mitochondria pre-rRNA processing may be mediated by other factors required for mitochondrial RNA processing. It is known that mitochondrial pre-rRNA processing is preceded by processing of tRNAs and this is mediated by mitochondrial RNase P, a protein complex that is composed of three subunits (MRPP1, MRPP2, and MRPP3) and is responsible for tRNA 5′ processing [52], [53]. Consistent with previous reports [53], siRNA-mediated reduction of MRPP1 led to accumulation of mitochondrial pre-rRNA, similar to the effects observed for P32 or RNase H1 reduction, as determined by northern hybridization (Figure 7A and 7B). Importantly, P32 was co-precipitated with MRPP1, but not with a control protein, RPL5, as demonstrated by immunoprecipitation followed by western analysis (Figure 7C), suggesting a physical link between P32 and the RNase P complex. Although we failed to detect co-precipitation of RNase H1 with MRPP1, even for RNase H1 over-expressed cells (data not shown), we cannot rule out the possibility that RNase H1 may also be linked to the RNase P complex by P32, given that the RNase H1 expression level is very low, or the interaction, if any, may be transient in nature.


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 can be co-immunoprecipitated with mitochondrial RNase P protein.(A) RT-PCR assay for the mRNA levels of P32 or MRPP1 in siRNA treated HeLa cells. The error bars represent standard deviation of three replicates. (B) Northern hybridization for mitochondrial pre-rRNA. Total RNA prepared from HeLa cells pre-treated for 24 hrs with P32 [(−)P32] or MRPP1 [(−)MRPP1] siRNAs was analyzed by northern hybridization, using probes specific to pre-rRNA, as in Figure 5.The same blot was re-probed for 7 SL RNA, which served as a loading control. (C) Immunoprecipitation using MRPP1 antibody (MRPP1-AB) or RPL5 antibody (RPL5-AB) was performed as described in materials and methods. Co-isolated proteins were separated by SDS-PAGE, and P32 protein was determined by western analysis. (−)AB, control immunoprecipitation in the absence of antibody. (D) DNase I treatment disrupted the P32-MRPP1 interaction. Immunoprecipitation was performed using MRPP1 antibody, as in panel C. After wash, the beads were incubated with either RNase A or DNase I, to elute bound materials. The eluted proteins were analyzed by western blots for the presence of P32 protein. Buffer, 1×TE buffer alone.
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pone-0071006-g007: P32 can be co-immunoprecipitated with mitochondrial RNase P protein.(A) RT-PCR assay for the mRNA levels of P32 or MRPP1 in siRNA treated HeLa cells. The error bars represent standard deviation of three replicates. (B) Northern hybridization for mitochondrial pre-rRNA. Total RNA prepared from HeLa cells pre-treated for 24 hrs with P32 [(−)P32] or MRPP1 [(−)MRPP1] siRNAs was analyzed by northern hybridization, using probes specific to pre-rRNA, as in Figure 5.The same blot was re-probed for 7 SL RNA, which served as a loading control. (C) Immunoprecipitation using MRPP1 antibody (MRPP1-AB) or RPL5 antibody (RPL5-AB) was performed as described in materials and methods. Co-isolated proteins were separated by SDS-PAGE, and P32 protein was determined by western analysis. (−)AB, control immunoprecipitation in the absence of antibody. (D) DNase I treatment disrupted the P32-MRPP1 interaction. Immunoprecipitation was performed using MRPP1 antibody, as in panel C. After wash, the beads were incubated with either RNase A or DNase I, to elute bound materials. The eluted proteins were analyzed by western blots for the presence of P32 protein. Buffer, 1×TE buffer alone.
Mentions: Since P32 itself does not contain nuclease domains and RNase H1 acts on DNA/RNA duplexes, the involvement of P32 and RNase H1 in mitochondria pre-rRNA processing may be mediated by other factors required for mitochondrial RNA processing. It is known that mitochondrial pre-rRNA processing is preceded by processing of tRNAs and this is mediated by mitochondrial RNase P, a protein complex that is composed of three subunits (MRPP1, MRPP2, and MRPP3) and is responsible for tRNA 5′ processing [52], [53]. Consistent with previous reports [53], siRNA-mediated reduction of MRPP1 led to accumulation of mitochondrial pre-rRNA, similar to the effects observed for P32 or RNase H1 reduction, as determined by northern hybridization (Figure 7A and 7B). Importantly, P32 was co-precipitated with MRPP1, but not with a control protein, RPL5, as demonstrated by immunoprecipitation followed by western analysis (Figure 7C), suggesting a physical link between P32 and the RNase P complex. Although we failed to detect co-precipitation of RNase H1 with MRPP1, even for RNase H1 over-expressed cells (data not shown), we cannot rule out the possibility that RNase H1 may also be linked to the RNase P complex by P32, given that the RNase H1 expression level is very low, or the interaction, if any, may be transient in nature.

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