RIBONUCLEASE P: Unity and Diversity in a tRNA Processing Ribozyme

Abstract

Ribonuclease P (RNase P) is the endoribonuclease that generates the mature 5 -ends of tRNA by removal of the 5 -leader elements of precursor-tRNAs. This enzyme has been characterized from representatives of all three domains of life (Archaea, Bacteria, and Eucarya) (1) as well as from mitochondria and chloroplasts. The cellular and mitochondrial RNase Ps are ribonucleoproteins, whereas the most extensively studied chloroplast RNase P (from spinach) is composed solely of protein. Remarkably, the RNA subunit of bacterial RNase P is catalytically active in vitro in the absence of the protein subunit (2). Although RNA-only activity has not been demonstrated for the archaeal, eucaryal, or mitochondrial RNAs, comparative sequence analysis has established that these RNAs are homologous (of common ancestry) to bacterial RNA. RNase P holoenzymes vary greatly in organizational complexity across the phylogenetic domains, primarily because of differences in the RNase P protein subunits: Mitochondrial, archaeal, and eucaryal holoenzymes contain larger, and perhaps more numerous, protein subunits than do the bacterial holoenzymes. However, that the nonbacterial RNase P RNAs retain significant structural similarity to their catalytically active bacterial counterparts indicates that the RNA remains the catalytic center of the enzyme. CONTENTS

Human ribonuclease P: Subunits, function, and intranuclear localization

“…Extensive purification analysis of RNase P from HeLa cells has revealed that the nuclear form of this tRNA processing holoenzyme is composed of at least 10 protein subunits associated with a single RNA species, H1 RNA (Table 1; Eder et al+, 1997;)+ These protein subunits are designated Rpp14, Rpp20, Rpp21, Rpp25, Rpp29, Rpp30, Rpp38, Rpp40, hPop5, and hPop1 (Lygerou et al+, 1996b;Eder et al+, 1997;Jarrous et al+, 1998Jarrous et al+, , 1999avan Eenennaam et al+, 1999van Eenennaam et al+, , 2001)+ The tight association of these proteins with highly purified nuclear RNase P obtained by different purification schemes implies that they constitute the core structure of the holoenzyme )+ A recent study has shown that the mitochondrial form of HeLa RNase P possesses an RNA that is identical to the H1 RNA (Puranam & Attardi, 2001)+ This enzyme has a sedimentation coefficient of ;17S in glycerol gradients, compared to ;15S of the nuclear counterpart, and exhibits properties typical of a ribonucleoprotein enzyme (Puranam & Attardi, 2001)+ This finding contradicts earlier biochemical analyses that support the concept that human mitochondrial RNase P is not a ribonucleoprotein complex (Rossmanith et al+, 1995;Rossmanith & Karwan, 1998)+ These opposing findings provoke further debate on the biochemical nature of mitochondrial and other organellar RNase P enzymes (Frank & Pace, 1998;Schon, 1999;Altman et al+, 2000;Gegenheimer, 2000;Salavati et al+, 2001), and therefore more biochemical and genetic means should be applied to resolve this issue+ Nuclear RNase P purified from Saccharomyces cerevisiae has nine distinct protein subunits and genetic studies established that these subunits are essential for yeast viability and enzyme activity in tRNA processing (see Xiao et al+, 2001)+ A multi-subunit ribonucleoprotein has also been described for nuclear RNase P purified from Aspergillus nidulans (Han et al+, 1998)+ In addition to the protein subunits described above (Table 1), several other proteins transiently interact with RNase P in yeast and human cells+ As judged by twohybrid genetic screens in yeast, the subunit Rpp20 interacts with the heat shock protein Hsp27 and the subunit Rpp14 contacts several proteins, including the LIM domain protein 1 (LIMD1) and HSPC232 ; the latter is an SR-rich protein that exhibits partial similarity to splicing factors SC-35 and SRp46+ The interaction of Rpp20 with Hsp27 was verified by biochemical analysis )+ In S. cerevisiae, a complex of seven Sm-like proteins (Lsm2-8) is associated with the precursor RNA subunit, Rpr1, of nuclear RNase ...…”

Dual function of the tRNA(m5U54)methyltransferase in tRNA maturation

“…Eukaryotic transfer RNA genes are transcribed by RNA polymerase III generating precursor tRNAs that have to be further processed to generate functional tRNAs (for review, see Wolin & Matera, 1999)+ One of the first proteins that bind the newly synthesized pre-tRNA is the La protein (Lhp1p in yeast), which binds to the 39 end of the transcript and protects this end from exonucleolytic digestion (Yoo & Wolin, 1997;Fan et al+, 1998)+ This is followed by removal of the 59 leader by RNase P (for review, see Frank & Pace, 1998), which precedes an endonucleolytic removal of the 39 trailer+ Furthermore, eukaryotic tRNAs have to undergo posttranscriptional addition of CCA to their 39 termini catalyzed by the tRNA nucleotidyltransferase (for review, see Deutscher, 1990)+ Some tRNA genes contain an intron and splicing of the pre-tRNA occurs before or after end maturation+ However, in Saccharomyces cerevisiae, end maturation normally precedes splicing (O'Connor & Peebles, 1991)+ During the maturation of tRNA, a variety of different nucleoside modifications occurs that allows the mature tRNA to function with high efficiency and flexibility+ Modified nucleosides can be found in all phylogenetic domains and also in identical positions of the tRNA, suggesting a conserved function of some tRNA modifications (Björk, 1986;Cermakian & Cedergren, 1998;Björk et al+, 2001)+ A 5-methyluridine residue at position 54 (m 5 U 54 ) is a highly conserved feature of eukaryotic and bacterial tRNAs+ The presence of this modification influences, in vitro, the fidelity and rate of protein synthesis as well as the stability of tRNA tertiary structure (Davanloo et al+, 1979;Kersten et al+, 1981)+ The in vivo role has been more difficult to elucidate+ An Escherichia coli strain with a mutation in the gene (trmA) encoding the tRNA(m 5 U 54 )methyltransferase and that lacks the m 5 U 54 nucleoside show a slight reduction in growth rate (Björk, 1986)+ However, a truncation of the trmA gene was shown to be lethal, indicative of an additional and essential function of the protein (Persson et al+, 1992)+ In contrast, an S. cerevisiae strain with either a mutation in or a deletion of the TRM2 gene, encoding the yeast tRNA(m 5 U 54 )methyltransferase, lacks the m 5 U 54 nucleoside but is viable and exhibits no apparent phenotype (Hopper et al+, 1982;Nordlund et al+, 2000)+ The methylation of U 54 is an early event in tRNA maturation in S. cerevisiae, as intron-containing pre-tRNAs have been shown to contain the m 5 U nucleoside (Knapp et al+, 1978;Etcheverry et al+, 1979)+ In this study, we report that yeast strains with each of four different mutant alleles of the single copy and essential sup61 ϩ gene, encoding tRNA CGA Ser , have a requirement for the TRM2 gene for growth at 30 8C+ The absence of the Trm2 protein ...…”

The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits

“…The holoenzyme of a bacterial RNase P is a ribonucleoprotein complex containing an RNA component (P RNA) of ;330-420 nt and a protein component (P protein) of ;120 amino acids (Frank & Pace, 1998;Altman & Kirsebom, 1999)+ The functional role of the protein component in the RNase P holoenzyme has been investigated extensively (Guerrier-Takada et al+, 1983, 1984Gardiner et al+, 1985;McClain et al+, 1987;Reich et al+, 1988;Peck-Miller & Altman, 1991;Svard & Kirsebom, 1992;Tallsjo & Kirsebom, 1993;Liu & Altman, 1994;Crary et al+, 1998;Kurz et al+, 1998;Loria et al+, 1998;Niranjanakumari et al+, 1998aNiranjanakumari et al+, , 1998bLoria & Pan, 1999)+ The Bacillus subtilis RNase P holoenzyme is remarkably efficient in the catalysis of precursor tRNA substrates with a k cat /K m near the diffusion limit (Kurz et al+, 1998;Reich et al+, 1988)+ In the absence of the protein component, k cat /K m decreases by 10 4 -fold under physiological conditions (Kurz et al+, 1998)+ A principal effect of the P protein function has been postulated to be the enhancement of substrate binding under physiological conditions (Crary et al+, 1998;Kurz et al+, 1998;Niranjanakumari et al+, 1998b)+ The physical state of the RNase P holoenzyme has received less attention+ Previous work by the Altman group conclusively showed that the Escherichia coli holoenzyme has an equal molar amount of RNA and protein (Talbot & Altman, 1994a)+ The affinity of the P protein binding to P RNA has been estimated to be ;0+5 nM, assuming a simple two-component binding isotherm (Talbot & Altman, 1994b)+ Functional studies by the Fierke group showed that the RNA-protein stoichiometry of the B. subtilis holoenzyme is also 1:1 (Niranjanakumari et al+, 1998a)+ Most studies in this area have focused on the details of P RNA-P protein interactions using chemical modification (Vioque et al+, 1988;Talbot & Altman, 1994b;Loria et al+, 1998;Biswas et al+, 20...…”