There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis. Received May 21, 1998; revised version accepted June 23, 1998. The physical organization of transcription and RNA processing events in eukaryotic nuclei has been studied extensively in the cases of pre-rRNAs and pre-mRNAs. Different stages in the expression of these RNAs are often associated with distinctive subnuclear structures, and these associations are integral to the synthesis and processing events. There is no direct information on the sites of tRNA gene transcription, although it is generally assumed that tRNA synthesis will be distributed throughout the nucleus; unlike the tandemly repeated ribosomal genes, the tRNA genes are not usually clustered in the chromosomes. In the budding yeast Saccharomyces cerevisiae, the 275 tRNA genes are scattered throughout the linear genome map (Cherry et al. 1997). Information on the sites of the numerous pre-tRNA processing events in the nucleus is also limited and has not provided a clear model for localization of the pathway.Maturation of eukaryotic pre-tRNA primary transcripts is often an ordered process, with 5Ј-and 3Ј-terminal processing preceding splicing (Tobian et al. 1985;Lee et al. 1991) and intron removal in turn preceding exit from nucleus to cytoplasm. In addition, multiple nucleotide modifications occur in the nucleus (Etcheverry et al. 1979;Hopper and Martin 1992). Although the order of processing events is not uniform for all pre-tRNAs and is not absolutely obligatory even for individual pre-tRNAs, the ordering suggests possible spatial organization of the maturation pathway. In yeast, current information on the sites of pre-tRNA processing could be consistent with either a dispersed pathway or one that is highly localized, especially to some portion of the nuclear periphery. Immunoelectron microscopy showed at least some of the tRNA splicing ligase to be near nuclear pores, leading to the speculation that the late splicing events in the nuclear pathway occur near the time that intron-containing pre-tRNAs exit to the cytoplasm (Clark and Abelson 1987). For intron-containing pretRNAs, it is therefore assumed that most of the nuclear precursor will retain the introns and that earlier processing events, such as removal of the termini, will need to take the introns into account (Leontis et al. 1988). Immunofluorescence microscopy has also localized a base modification enzyme, N 2 ,N 2 -dimethylguanosine-s...

Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5 termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two-and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific proteinprotein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae. Ribonuclease P (RNase P) is an essential endoribonuclease that acts early in tRNA biogenesis to remove the 5Ј leader sequences of precursor tRNAs (pretRNAs) (1-3). The enzyme has been identified in every organism tested, in all kingdoms of life. In most cases, the enzyme is composed of a single RNA subunit and one or more protein subunits (1, 4). The RNA subunit forms the catalytic core of the enzyme, and the bacterial and some archaeal RNA subunits alone are catalytic in vitro (5-7). In contrast, no eukaryotic RNase P RNA subunits have been shown to be catalytic in the absence of protein. In bacteria, RNase P is composed of a catalytic RNA subunit and a single small protein subunit. Studies on the bacterial RNase P protein suggest that the protein plays a role in substrate recognition (8)(9)(10)(11). Recent data show that at least one form of archaeal RNase P consists of four or more proteins and a single RNA subunit (12). Moreover, the identified archaeal proteins appear to be homologs of the eukaryotic RNase P proteins and not the bacterial proteins (T. A. Hall and J. W. Brown, personal communication).Eukaryotic nuclear RNase P contains an RNA subunit similar in size to its bacterial and archaeal counterparts, containing all of the most conserved ''critical regions'' from the bacterial consensus structure (13). However, the protein content is far more complex and is absolutely required for activity. Human nuclear RNase P appears to contain at least ten proteins (14-19). At le...

The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements.

Ribonuclease P is an ancient enzyme that cleaves pre-tRNAs to generate mature 5' ends. It contains an essential RNA subunit in Bacteria, Archaea, and Eukarya, but the degree to which the RNA subunit relies on proteins to supplement catalysis is highly variable. The eukaryotic nuclear holoenzyme has recently been found to contain almost twenty times the protein content of the bacterial enzymes, in addition to having split into at least two related enzymes with distinct substrate specificity. In this review, recent progress in understanding the molecular architecture and functions of nuclear forms of RNase P will be considered.