Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

Hongjiang Wu,Walt F Lima,Stanley T Crooke,Hong Sun,Xuehai Liang,Janine Santos

doi:10.1371/journal.pone.0071006

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.

Highlights

Ribonuclease H hydrolyzes the RNA strand in RNA-DNA hybrids [1]
H1 and its roles in the activity of antisense oligonucleotidedirected substrate RNA degradation, we identified RNase H1associated proteins by co-immunoprecipitation from cell lines that stably express epitope-tagged human RNase H1 and H2, using antibodies specific to the tags
Proteins associated with RNase H1 or RNase H2 were co-immunoprecipitated from extracts of cells that stably express the tagged proteins, using either anti-Flag or anti-HA beads (Figure 1B)

Summary

Introduction

RNase H activity appears to be ubiquitous in most organisms [2,3,4,5,6,7,8]. RNases H constitute a family of proteins with different molecular masses, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. Strong amino acid sequence homology was observed for type 1 RNase H (RNase H1) from human, yeast, chicken, E. coli and mouse. Type 2 RNase H (RNase H2) from human, Caenorhabditis elegans, yeast and E. coli. Exhibit strong sequence homology [11] Both enzymes have been shown to be expressed ubiquitously in all cells and tissues. RNase H1 enzymes hydrolyze RNA/DNA hybrids containing at least four ribonucleotides [12], while type 2 enzymes can hydrolyze DNA-RNA containing a single ribonucleotide [7,12,13,14]

Methods

Results

Conclusion