Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P

Abstract

Ribonuclease P is the enzyme responsible for removing the 59-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-R P nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-R P oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA-or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-R P oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-R P oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

“…RNase P is primarily responsible for the maturation of 5 ′ -ends of tRNA (for review, see Evans et al 2006;Marvin and Engelke 2009;Altman 2010;Esakova and Krasilnikov 2010;Jarrous and Gopalan 2010). The phylogenetically conserved RNA component of RNase P is the catalytic moiety of the enzyme (Guerrier-Takada et al 1983;Chen and Pace 1997;Pannucci et al 1999;Thomas et al 2000;Kikovska et al 2007;). In a number of cases, the ribonucleoprotein RNase P is supplemented or altogether replaced by unrelated protein-only enzymes termed PRORPs (Pinker et al 2013).…”

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

“…RNase P is universally responsible for the maturation of the 59 end of tRNA and is involved in the metabolism of a variety of other RNA molecules (Coughlin et al 2008;Altman 2010;Marvin et al 2011a); in addition, human RNase P was suggested to play a role in transcription (Reiner et al 2006(Reiner et al , 2008. The well-conserved RNA component of RNase P (Chen and Pace 1997) is the catalytic subunit of the enzyme in all domains of life (Guerrier-Takada et al 1983;Pannucci et al 1999;Thomas et al 2000;Kikovska et al 2007;). The protein composition of RNase P varies: While bacterial enzymes have a single small protein (Stark et al 1978), archaeal RNases P have four to five proteins (Hall and Brown 2002;Cho et al 2010), and eukaryotic RNases P contain a large multicomponent protein part (nine essential proteins in Saccharomyces cerevisiae; for review, see Marvin and Engelke 2009;Esakova and Krasilnikov 2010).…”

Substrate recognition by ribonucleoprotein ribonuclease MRP

“…The RNA component of RNase P (Fig. 1A) is the enzyme's catalytic moiety (Guerrier-Takada et al 1983;Pannucci et al 1999;Thomas et al 2000;Kikovska et al 2007); the RNA component of RNase MRP (Fig. 1B) shows clear similarity to that of RNase P. In particular, the catalytic (C-) domain of RNase P (a generally phylogenetically conserved domain that contains the active site) appears to have the same overall architecture as the corresponding part of RNase MRP (Domain 1, Fig.…”