The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors.

Basic peptides covalently linked to nucleic acids, or chemically modified nucleic acids, enable the insertion of such a conjugate into bacteria grown in liquid medium and mammalian cells in tissue culture. A unique peptide, derived from human T cells, has been employed in a chemical synthesis to make a conjugate with a morpholino oligonucleotide. This new conjugate is at least 10- to 100-fold more effective than previous peptides used in altering the phenotype of host bacteria if the external guide sequence methodology is employed in these experiments. Bacteria with target genes expressing chloramphenicol resistance, penicillin resistance, or gyrase A function can effectively be reduced in their expression and the host cells killed. Several bacteria are susceptible to this treatment, which has a broad range of potency. The loss in viability of bacteria is not due only to complementarity with a target RNA and the action of RNase P, but also to a non-gene-specific tight binding of the complexed nontargeted RNA to the basic polypeptide-morpholino oligonucleotide.

Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P. Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA. Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels. Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme. Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P.

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The expression of gene products in bacteria can be inhibited by the use of RNA external guide sequences (EGSs) that hybridize to a target mRNA. Endogenous RNase P cleaves the mRNA in the complex, making it inactive. EGSs participate in this biochemical reaction as the data presented here show. They promote mRNA cleavage at the expected site and sometimes at other secondary sites. Higher-order structure must affect these reactions if the cleavage does not occur at the defined site, which has been determined by techniques based on their ability to find sites that are accessible to the EGS oligonucleotides. Sites defined by a random EGS technique occur as expected. Oligonucleotides made up primarily of defined or random nucleotides are extremely useful in inhibiting expression of the gyrA and rnpA genes from several different bacteria or the cat gene that determines resistance to chloramphenicol in Escherichia coli. An EGS made up of a peptidephosphorodiamidate morpholino oligonucleotide (PPMO) does not cleave at the same site as an unmodified RNA EGS for reasons that are only partly understood. However, PPMO-EGSs are useful in inhibiting the expression of targeted genes from Gram-negative and Gram-positive organisms during ordinary growth in broth and may provide a basis for broad-spectrum antibiotics.drug resistance ͉ Gram-positive and Gram-negative bacteria ͉ peptide-phosphorodiamidate morpholino oligonucleotide ͉ RNase P T he utility of bacterial transformation for therapeutic purposes has been limited by the number of species that will undergo transformation and the frequency with which that event happens. To accommodate new therapies that involve small nucleic acids, a means has to be developed to enable bacterial species to take up these nucleic acids with relative ease. The covalent linkage of arginine-rich peptides to the ends of chemically-modified RNAs facilitates the uptake of the RNA analog (1, 2) and other similar molecules (4,5). This methodology in combination with an effective means of inactivating gene expression has to be developed to make it useful for therapeutic agents. There are other processes that function in bacteria to inhibit gene expression (3, 6), but the external guide sequence (EGS) technology (7,8) that is mediated by RNase P cleavage of the target RNA seems optimal in this regard.RNAi and siRNA (ref. 9 and references therein) are not useful tools for the transformation of bacterial species because these RNAs rely on an intracellular complex, the Dicer complex (9) in particular, to release ssRNA that will base-pair with the target mRNA. EGS technology, which is just as effective as siRNA in mammalian cells in tissue culture (10), is very effective in Escherichia coli (11,12) and Salmonella typhimurium (13). Bacterial cells can be altered from drug resistance to drug sensitivity with the methods generally described here (11), and a similar method has also been reported (14). Essential genes can also be inactivated in terms of their expression. The EGS method will allow 3-bp mismatche...

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.RNase P ͉ tRNA processing C urrent antimicrobial drugs inhibit bacteria primarily by targeting essential proteins (or protein-mediated processes) conserved among many bacterial species (1). Accordingly, medical and agricultural antimicrobials not only treat pathogenic bacterial infections, but also affect commensal bacteria. This broad spectrum of activity creates side effects for individual patients (2, 3) and also exerts selective pressure for the emergence, spread, and interspecies transfer of resistance (4, 5). Here, we report antimicrobial activity mediated by using techniques of external guide sequences (EGSs) that enhance RNase Pmediated mRNA cleavage (6, 7). We inhibited bacterial growth by reducing the level of expression for two different essential proteins that exist in fewer than 1,500 copies per Escherichia coli, RNase P C5 protein (8), and gyrase A (9).RNase P catalyzes tRNA processing (10). The tRNA products of these cleavages result in important components of the protein synthetic mechanisms. This enzyme contains a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) in E. coli. Gyrase A catalyzes chromosomal DNA supercoiling during replication (11), is the molecular target of quinolone antimicrobials (12), and can mediate quinolone resistance (13-15). Eukaryotes also express essential RNase P subunits (10) and gyrase (16) enzymes (the later a target for antineoplastic agents), but they are quite distinct from their bacterial homologs in RNA and protein sequence (17) as well as in some aspects of enzymatic reaction details (10, 16).E. coli EGSs were based on species-specific gyrase A sequences that are not present in Salmonella typhimurium, but which encode identical gyrase A protein sequences in both species (15,18), and the C5 protein subunit of RNase P. This system provides a foundation for general strategies to attack bacteria and a tool for the inducible disruption of bacterial gene products. It also suggests mechanisms for a degree of exquisitely narrow spectrum antimicrobial activity, discriminating between and selectively inhibiting even closely related species of bacteria based on fine differences in bacterial coding sequences.The inhibition of bacterial growth via EGS-targeted technology exhibits species specificity and dose-response. The cleavage is independent of certain mutations of up to three bases in the gyra...

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5 UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5 UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.arginase ͉ Aspergillus nidulans ͉ Bacillus subtilis ͉ coenzyme B12 ͉ Escherichia coli R iboswitches occur in the untranslated regions at the 5Ј side of the mRNA for translated genes (1). In some cases, the riboswitch extends into the ORF of the gene to be transcribed and translated. Small metabolites that are metabolized or transported by the products of the ORFs downstream from the riboswitch interact with the riboswitch without the action of proteins and control the translation, and possibly the transcription, of the riboswitch and the ORF. These results indicate the general importance and antiquity of RNA-based methods in gene regulation.In this paper, we show that RNase P (2) cleaves some riboswitches once. In fact, we show this for the riboswitch in the 5Ј region of the btuB gene in Escherichia coli and a similar gene in Bacillus subtilis (3). The putative arginine riboswitch, the 5Ј region before the arginase gene in Aspergillus nidulans (4) is also cleaved, indicating that the unusual RNase P cleavage activity is a phenomenon that is widespread in eukaryotes as well as in prokaryotes. The RNase P cleavage occurs actually in the beginning of the gene coding for the arginase gene. We also assayed four other riboswitches found in E. coli but found no cleavage by RNase P in those regions.Some genetic experiments indicate that in E. coli, the presence of RNase P cleavage in the btuB riboswitch might promote degradation of the 3Ј region downstream from the cleavage site. In operons, this result is a polarity effect and means that the downstream genes after the enzyme cleavage site are usually degraded quickly, and the ORF is not translated as frequently as the upstream genes (5). A polarity effect in riboswitches means a loss of the uncleaved translation efficiency of the downstream gene. It is also certainly the case that RNase P does cleave some substrates that are not operons or mature small stable RNA precursors (2, 6), but the biological function of these latter cleavages is not fully understood.Alt...

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.