Catalytic RNA Component Research Articles

Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters.

RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occupancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings reshape our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.

Proteins Rpr2 and Pop3 increase the activity and thermal stability of yeast RNase P

ABSTRACT RNA-based enzyme RNase P is a ribonucleoprotein complex responsible primarily for 5’-maturation of tRNAs. S. cerevisiae RNase P comprises a catalytic RNA component and nine proteins. The assembly and maturation of S. cerevisiae RNase P involves an abundant and catalytically active precursor form, which includes all components except for proteins Rpr2 and Pop3. Rpr2 and Pop3 are essential proteins, but their roles in RNase P were not clear. Here we use a step-wise in vitro assembly of yeast RNase P to show that the addition of proteins Rpr2 and Pop3 increases the activity and thermal stability of the RNase P complex, similar to the effects previously observed for archaeal RNases P.

Telomerase Inhibitors from Natural Products and Their Anticancer Potential.

Telomeres and telomerase are nowadays exploring traits on targets for anticancer therapy. Telomerase is a unique reverse transcriptase enzyme, considered as a primary factor in almost all cancer cells, which is mainly responsible to regulate the telomere length. Hence, telomerase ensures the indefinite cell proliferation during malignancy—a hallmark of cancer—and this distinctive feature has provided telomerase as the preferred target for drug development in cancer therapy. Deactivation of telomerase and telomere destabilization by natural products provides an opening to succeed new targets for cancer therapy. This review aims to provide a fundamental knowledge for research on telomere, working regulation of telomerase and its various binding proteins to inhibit the telomere/telomerase complex. In addition, the review summarizes the inhibitors of the enzyme catalytic subunit and RNA component, natural products that target telomeres, and suppression of transcriptional and post-transcriptional levels. This extensive understanding of telomerase biology will provide indispensable information for enhancing the efficiency of rational anti-cancer drug design.

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

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA–RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P.

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5' end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.

By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version — whether defined at the sequence, structure or codon usage level — does not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification.

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

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

Expressional Profiling of Telomerase and Telomere-Associated Molecules in the Rat Testis and Seminal Vesicle during Postnatal Developmental Period

Maintenance of adequate telomere length in developing cells is the most important concern to preserve the integrity of the genome. The length of telomere is strictly regulated by numerous telomere-binding proteins and/or interacting factors. Even though the expression of telomerase in the male reproductive tract has been characterized, developmental expressional profiling of telomerase and other telomere-associated proteins has not been determined in detail. The present study was attempted to examine expression patterns of catalytic subunit (Tert) and RNA component (Terc) of telomerase and two telomerase associated factors, telomerase associated protein 1 (Tep1) and TERF1 (TRF1) interacting nuclear factor 2 (Tinf2) in the testis and seminal vesicle of male rat during postnatal development. The real-time PCR analysis was utilized to quantify mRNA expression of molecules. The abundance of Tep1 mRNA in the testis and seminal vesicle was the highest at 5 months of age. Expressional fluctuation of Tinf2 during postnatal development was found in the testis, while expression of Tinf2 in the seminal vesicle was gradually increased until 5 months of age and then significantly decreased later. mRNA level of Tert gene in the testis was significantly increased at the adult and the elder, while the highest expression of Tert gene in the seminal vesicle was found at 5 months of age. Expression of Terc transcript in the testis and seminal vesicle was the highest at 5 months of age, followed by significant reduction at 1 and 2 years of ages. Such differential gene expression of telomere-associated factors and telomerase components in different male reproductive tissues during postnatal development indicates that maintenance of telomere length would be regulated in tissue- and/or age-specific manners. (Key words : Telomerase, Testis, Seminal vesicle, Development, Telomerase-associated molecule)

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain

Ribonuclease (RNase) P is a site-specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix-loop-helix protein-binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 A. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.

Crystallization and preliminary X-ray diffraction analysis of the P3 RNA domain of yeast ribonuclease MRP in a complex with RNase P/MRP protein components Pop6 and Pop7.

Eukaryotic ribonucleases P and MRP are closely related RNA-based enzymes which contain a catalytic RNA component and several protein subunits. The roles of the protein subunits in the structure and function of eukaryotic ribonucleases P and MRP are not clear. Crystals of a complex that included a circularly permuted 46-nucleotide-long P3 domain of the RNA component of Saccharomyces cerevisiae ribonuclease MRP and selenomethionine derivatives of the shared ribonuclease P/MRP protein components Pop6 (18.2 kDa) and Pop7 (15.8 kDa) were obtained using the sitting-drop vapour-diffusion method. The crystals belonged to space group P4(2)22 (unit-cell parameters a = b = 127.2, c = 76.8 A, alpha = beta = gamma = 90 degrees ) and diffracted to 3.25 A resolution.

TRNA recognition, processing, and disease: Hypotheses around an unorthodox type of RNase P in human mitochondria

RNase P is the endonuclease responsible for the maturation of the 5′ ends of tRNAs. A catalytic RNA component was long considered the premier attribute of the enzyme family. Ignoring this heritage, human mitochondria make their RNase P of three proteins only. While one of them appears to be the metallonuclease actually responsible for phosphodiester hydrolysis, the other two have been recruited from unrelated biochemical pathways and may be critical for substrate recognition. One of them is moreover identical to a previously identified amyloid-β-binding protein, whereby it could link tRNA processing to mitochondrial dysfunction in Alzheimer’s disease.

Telomerase and its potential for therapeutic intervention.

Telomerase and telomeres are attractive targets for anticancer therapy. This is supported by the fact that the majority of human cancers express the enzyme telomerase which is essential to maintain their telomere length and thus, to ensure indefinite cell proliferation--a hallmark of cancer. Tumours have relatively shorter telomeres compared to normal cell types, opening the possibility that human cancers may be considerably more susceptible to killing by agents that inhibit telomere replication than normal cells. Advances in the understanding of the regulation of telomerase activity and the telomere structure, as well as the identification of telomerase and telomere associated binding proteins have opened new avenues for therapeutic intervention. Here, we review telomere and telomerase biology and the various approaches which have been developed to inhibit the telomere/telomerase complex over the past decade. They include inhibitors of the enzyme catalytic subunit and RNA component, agents that target telomeres, telomerase vaccines and drugs targeting binding proteins. The emerging role of telomerase in cancer stem cells and the implications for cancer therapy are also discussed.

Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells

Human telomerase is a ribonucleoprotein complex composed of at least the reverse catalytic transcriptase hTERT and RNA component hTR. The enzyme stabilizes telomere length and thereby contributes to unlimited cell proliferation, i.e. immortality. Reactivation of telomerase activity during carcinogenesis is a common hallmark in most human tumor types. Consequently, telomerase is an attractive molecular target toward which to direct cancer therapeutic agents. RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells by targeting with short duplex RNA (short-interfering RNA or siRNA). Thus, we evaluated the ability of siRNAs to inhibit telomerase activity in the HT29 immortal human colorectal adenocarcinoma cell line as a model for colorectal carcinogenesis. By transient expression of a specific siRNA directed against hTERT, we reduced telomerase activity in the transfected cells. Moreover, telomere lengths were reduced in cells stably expressing this particular RNA sequence, cloned as an shRNA into an eukaryotic expression vector. The cell clone that displayed a telomerase-negative phenotype showed dramatically reduced telomere lengths and stopped proliferation. We observed that the vector was integrated into the cell genome and, despite telomere shortening, cells retained their MSI phenotype. We conclude that we have developed a potent telomerase inhibitor leading to cell death.

Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA.

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme.

The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA

Ribonuclease P (RNaseP) catalyses the removal of the 5′-leader sequence from pre-tRNA to produce the mature 5′ terminus. The prokaryotic RNaseP holoenzyme consists of a catalytic RNA component and a protein subunit (RNaseP protein), which plays an auxiliary but essential role in vivo by binding to the 5′-leader sequence and broadening the substrate specificity of the ribozyme. We determined the three-dimensional high-resolution structure of the RNaseP protein from Staphylococcus aureus (117 amino acid residues) by nuclear magnetic resonance (NMR) spectroscopy in solution. The protein has an αβ-fold, similar to the ribonucleoprotein domain. We used small nucleic acid molecules as a model for the 5′-leader sequence to probe the propensity for generic single-stranded RNA binding on the protein surface. The NMR results reveal a contiguous interaction site, which is identical with the previously identified leader sequence binding site in RNaseP holoenzyme. The conserved arginine-rich motif does not bind single-stranded RNA. It is likely that this peptide segment binds selectively to double-stranded sections of P RNA, which are conformationally more rigid. Given the essentiality of RNaseP for the viability of the organism, knowledge of the S. aureus protein structure and insight into its interaction with RNA will help us to develop RNaseP and RNaseP protein as targets for novel antibiotics against this pathogen.

Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance

Antibiotic resistance is rapidly becoming a major medical problem. Many antibiotics are directed against bacterial ribosomes, and mutations within both the RNA and protein components can render them ineffective. It is well known that the majority of these antibiotics act by binding to the ribosomal RNA, and it is of interest to understand how mutations in the ribosomal proteins can produce resistance. Translational accuracy is one important target of antibiotics, and a number of ribosomal protein mutations in Escherichia coli are known to modulate the proofreading mechanism of the ribosome. Here we describe the high-resolution structures of two such ribosomal proteins and characterize these mutations. The S5 protein, from the small ribosomal unit, is associated with two types of mutations: those that reduce translational fidelity and others that produce resistance to the antibiotic spectinomycin. The L6 protein, from the large subunit, has mutations that cause resistance to several aminoglycoside antibiotics, notably gentamicin. In both proteins, the mutations occur within their putative RNA-binding sites. The L6 mutations are particularly drastic because they result in large deletions of an RNA-binding region. These results support the hypothesis that the mutations create local distortions of the catalytic RNA component. When combined with a variety of structural and biochemical data, these mutations also become important probes of the architecture and function of the translational machinery. We propose that the C-terminal half of S5, which contains the accuracy mutations, organizes RNA structures associated with the decoding region, and the N-terminal half, which contains the spectinomycin-resistance mutations, directly interacts with an RNA helix that binds this antibiotic. As regards L6, we suggest that the mutations indirectly affect proofreading by locally distorting the EF-Tu·GTP·aminoacyl tRNA binding site on the large subunit.

Sequence and functional characterization of RNase P RNA from the chl a/b containing cyanobacterium Prochlorothrix hollandica

Only a few complete sequences and very limited functional data are available for the catalytic RNA component of cyanobacterial RNase P. The RNase P RNA from the chl a/ b containing cyanobacterium Prochlorothrix hollandica belongs to a rarely found structural subtype with an extended P15/16 domain. We have established conditions for optimal in vitro ribozyme activity, and determined the kinetic parameters for cleavage of pre-tRNA Tyr. Analysis of pre-tRNA mutants revealed that the T-stem sequence only plays a modulating role, whereas the CCA end is essential for efficient product formation.

The catalytic core of RNase P.

A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA.

Comparative Analysis of Ribonuclease P RNA Structure in Archaea

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.

Characterization of ribonuclease P RNAs from thermophilic bacteria.

The catalytic RNA component of bacterial RNase P is responsible for the removal of 5' leader sequences from precursor tRNAs. As part of an on-going phylogenetic comparative characterization of bacterial RNase P, the genes encoding RNase P RNA from the thermophiles Thermotoga maritima, Thermotoga neapolitana, Thermus aquaticus, and a mesophilic relative of the latter, Deinococcus radiodurans, have been cloned and sequenced. RNAs transcribed from these genes in vitro are catalytically active in the absence of other components. Active holoenzymes have been reconstituted from the T.aquaticus and T.maritima RNAs and the protein component of RNase P from Escherichia coli. The RNase P RNAs of T.aquaticus and T.martima, synthesized in vitro, were characterized biochemically and shown to be inherently resistant to thermal disruption. Several features of these RNAs suggest mechanisms contributing to thermostability. The new sequences provide correlations that refine the secondary structure model of bacterial RNase P RNA.