[9] - Human Ribonuclease P

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Ribonuclease P is a ribonucleoprotein nuclease required for the site-specific cleavage of the 5′ leader sequence of precursor tRNAs. In eubacteria, the RNA subunit of RNase P is the catalytic moiety and is capable of processing precursor tRNA in the presence of divalent metal ions. The RNA subunits of eukaryotic RNase P (human and yeast) do not exhibit enzymatic activity In Vitro, an indication that their protein components are required for the catalytic reaction. This chapter describes a purification procedure for human RNase P from HeLa cells that facilitated the characterization of nine of its protein subunits. This procedure also allows the separation of RNase P from the structurally related RNase MRP nuclease. The purification procedure shows that nuclear RNase P from HeLa cells has at least nine protein subunits in association with a single RNA species, H1 RNA. Nuclear RNase P in other eukaryotes, such as S. cerevisiae and Aspergillus nidulans, also consists of multiple protein subunits, some of which are evolutionarily conserved and an RNA subunit. The precise function of these subunits in tRNA processing is not yet understood. Reconstitution of the activity of RNase P in vitro may reveal the role of these subunits in catalysis.

Introduction

Ribonuclease P is a ribonucleoprotein nuclease required for the site-specific cleavage of the 5′ leader sequence of precursor tRNAs. In eubacteria, the RNA subunit of RNase P is the catalytic moiety and is capable of processing precursor tRNA in the presence of divalent metal ions.1 The single protein subunit of bacterial RNase P acts as a cofactor.2, 3 In contrast, the RNA subunits of eukaryotic RNase P (human and yeast) do not exhibit enzymatic activity in vitro, an indication that their protein components are required for the catalytic reaction. In this article, we describe a purification procedure for human RNase P from HeLa cells that facilitated the characterization of nine of its protein subunits. This procedure also allows the separation of RNase P from the structurally related RNase MRP nuclease.

Section snippets

Preparation of S100 Extracts of HeLa S3 Cells

An outline of a purification procedure of human RNase P from HeLa cells is shown in Fig. 1. For purification of sufficient RNase P for analysis of protein subunits,4 frozen pellets of HeLa S3 cells [Cell Culture Center, National Center for Research Resources (NCRR), National Institutes of Health (NIH), Bethesda, MD] harvested from 120 liters of cell culture at a density of 6 × 105 cells/ml are used. The estimated copy number of RNase P is ~2 × 105/cell.5 Cell pellets are placed on ice for 1–2 hr for

Technical Considerations in Preparation and Storage of Active RNase P

Highly purified RNase P is an unstable complex, sensitive to both heat and to prolonged storage at 4°. Accordingly, storage of active enzyme is recommended in 30% (v/v) glycerol at ‒20°.

Because RNase P is a ribonucleoprotein, special caution must be undertaken during the purification procedure to avoid nuclease and protease contamination. In addition to the full-length 340-nucleotide H1 RNA, two smaller derivatives of H1 RNA of 160–170 nucleotides are detected in RNase P preparations as judged

Assay of RNase P Activity

The only reliable assay for measuring RNase P activity is a PAGE analysis of the processing of the 5′ leader sequence of precursor tRNA. The specific activity of RNase P is measured in units of enzyme per milligram protein (Table I). One unit of RNase P is defined as the amount of enzyme required to process 1 pmol of precursor tRNA in 1 min at 37° 4 A standard buffer for testing active human RNase P in processing of a 32P-labeled precursor tRNA is composed of 10 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 100

Characterization of Protein Subunits of RNase P

RNase P purified through the gel-filtration steps (main pathway in Fig. 1) described above was separated in a preparative protein gel, followed by Coomassie blue staining.4,12 Copurifying polypeptides (>100 ng) were excised from the gel and subjected to microsequencing of tryptic peptides (W. M. Keck Facility at Yale University)4,9 The peptide sequences obtained were used for searching sequence databases (GenBank and EMBL) for their corresponding cDNA clones (expressed sequence tags), or for

Conclusions

The purification procedure described above shows that nuclear RNase P from HeLa cells has at least nine protein subunits in association with a single RNA species, H1 RNA. Nuclear RNase P in other eukaryotes, such as S. cerevisiae15 and Aspergillus nidulans,17 also consists of multiple protein subunits, some of which are evolutionarily conserved,11,13,15 and an RNA subunit. The precise function of these subunits in tRNA processing is not yet understood. Reconstitution of the activity of RNase P

Acknowledgments

We thank Craig Crews (Yale University) and Dieter Söil (Yale University) for the use of FPLC apparatus and columns, and our colleagues Cecilia Guerrier-Takada and Donna Wesolowski for assistance and comments on this manuscript. This work was supported by U.S. Public Health Service Grant GM-19422 and Human Frontiers Science Program Grant RG 02N1 1997M to S. Altman. N. Jarrous is the recipient of a Kahanoff Foundation fellowship and supported by the Leszynski Fund for Advanced Research.

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