RNase P: Variations and Uses

Abstract

This essay will bring to date a picture of the properties of RNase P from several organisms and a summary of how this enzyme can be used to decrease specific gene expression. Current details of how the enzyme works and other features governing its reaction are reviewed elsewhere (1-3).RNase P is responsible for generating the mature 5Ј-end of tRNAs by a single endonucleolytic cleavage of their precursors. It is an essential, ubiquitous enzyme present in all cells and cellular compartments that synthesize tRNA: bacterial cells, eukaryotic nuclei, mitochondria, and chloroplasts. The essential function in vivo of RNase P has been demonstrated in those systems amenable to genetic analysis such as bacteria (4) and yeast nuclei (5) and mitochondria (6, 7). All known RNase P enzymes are ribonucleoproteins and contain an RNA subunit essential for catalysis with the possible exception of RNase P in some plant chloroplasts and trypanosome mitochondria (8, 9).The chemical mechanism of RNase P involves essential divalent metal ions (2) and is thought to be an in-line S N 2 displacement reaction (1). The endonucleolytic cleavage generates 5Ј-phosphate and 3Ј-hydroxyl end groups in the products. For our purposes, the way in which the enzyme recognizes substrates is an important feature of its ability to lower the amount of any particular RNA and expression inside cells. Natural substrates can be reduced to two oligonucleotides, which when hydrogen bonded together (Fig. 1) resemble sufficiently the essential features of a substrate so that one of the oligonucleotides, the target RNA (i.e. any RNA inside the cell), is cleaved efficiently by the enzyme and inactivated. This important aspect of RNase P revolves entirely around its substrate recognition mechanism and does not depend on the fact that there is an RNA subunit in the enzyme.The RNA Subunit of RNase P The RNA component of RNase P from bacteria is encoded by the rnpB gene and varies in length between about 350 and 450 nucleotides (10). There is little sequence similarity among the 300 or so bacterial sequences except for a few short segments. Phylogenetic covariation analysis of the large data set has allowed the precise definition of the secondary structure and the identification of several tertiary interactions (11)(12)(13)(14)(15). This RNA from bacteria can be divided into two distinct structural classes: type A, represented by Escherichia coli, which is the ancestral type found in most bacteria (the RNA subunit of the enzyme from E. coli is called M1 RNA and is referred to by that name herein), and type B, represented by Bacillus subtilis, which is found in the low GC content Grampositive bacteria (16). An intermediate structure (type C) is found in green non-sulfur bacteria (12). Despite differences in the secondary structure organization of type A and type B RNAs, both RNAs can be modeled into a similar three-dimensional structure with the evolutionarily conserved nucleotides placed in nearly identical positions (16). These computer-aided modeling efforts illustr...

Inhibition of no tail (ntl) gene expression in zebrafish by external guide sequence (EGS) technique

“…Site was chosen in looped regions to provide accessible regions in the target RNA. In addition, the acceptor and D-stem equivalent in 3/4 EGS or m3/4 EGS were designed to contain 7 and 4 bp complementary to the target RNA as described by Gopalan et al [4]. The targeting sites of ntl mRNA is shown in Fig.…”

“…This method has been successfully used to downregulate the expression of genes in bacteria, human and plant cells in tissue culture [1][2][3]. Further studies indicate that 3/4 EGS without anticode loop had the same function to cleave the target mRNA, which was termed minimized 3/4 EGS or m3/4 EGS [4]. Encouraged by these results, we tried to determinate the function of genes in zebrafish using EGS strategy.…”

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

“…Ribozymes exist as naturally occurring catalytic RNAs, expressed in a wide range of living organisms [5,6]. In this review, we focus on RNase P and its derivatives and highlight some of the most recent studies in utilizing RNase P for the development of nucleic acid-based gene therapy, with a special focus on antiviral applications.…”

“…First, the mechanism of the EGS technology for degradation of a specific mRNA is different from other RNA-or DNA-based gene-targeting approaches. It uses the endogenous RNase P, which is one of the most ubiquitous, abundant, stable and efficient enzymes in all type of cells [6,8,85]. This essential enzyme is highly expressed (5×10 4 copies per cell) and is responsible for the processing of all tRNA precursors that account for approximately 2% of total cellular RNA [6].…”

“…Our initial library of ribozymes contained random mutations in regions that are known to be conserved across all species of RNase P catalytic RNAs and are important for catalysis and substrate binding [66][67][68]. The in vitro evolution process was carried out in a series of steps that included (1) annealing of M1GS RNA pool with a 5′ biotinylated substrate, (2) binding the complex to streptavidin-agarose column, (3) M1GS RNA-mediated cleavage of the substrate in the presence of divalent ion-containing buffer, (4) recovering M1 RNA with denaturing gel electrophoresis, (5) synthesis of cDNA copies of RNA molecules with RT-PCR, followed by (6) in vitro transcription of the generated cDNAs with T7 RNA polymerase (Figure 2) [65,69,70]. The sequences isolated after several rounds of selection were cloned and determined.…”