Ribonuclease P (RNase P) is an essential endonuclease that acts early in the tRNA biogenesis pathway. This enzyme catalyzes cleavage of the leader sequence of precursor tRNAs (pre-tRNAs), generating the mature 5′ end of tRNAs. RNase P activities have been identified in Bacteria, Archaea, and Eucarya, as well as organelles. Most forms of RNase P are ribonucleoproteins, i.e. they consist of an essential RNA subunit and protein subunits, although the composition of the enzyme in mitochondria and chloroplasts is still under debate. The recent purification of the eukaryotic nuclear RNase P has demonstrated a significantly larger protein content compared to the bacterial enzyme. Moreover, emerging evidence suggests that the eukaryotic RNase P has evolved into at least two related nuclear enzymes with distinct functions, RNase P and RNase MRP. Here we review current information on RNase P, with emphasis on the composition, structure, and functions of the eukaryotic nuclear holoenzyme, and its relationship with RNase MRP.

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the threehybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.

SummaryIsolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using affinity purification methods that are specific for the RNA rather than the protein components of these complexes. We describe the use of two such RNA affinity tags: small RNAs that bind with high affinity and specificity to either Sephadex beads or streptavidin affinity resins and can be eluted under mild, native conditions that retain intact complexes. The tags can be inserted into appropriate locations in genes encoding the RNA components, and ribonucleoproteins can be assembled either in vivo or in vitro before affinity isolation. Strategies toward the design and production of these tagged RNA sequences are discussed, and the purification procedure is outlined.

The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.