A protein-only RNase P in human mitochondria.

In bacteria, archaea, and the eukaryote nucleus, the endonuclease ribonuclease P (RNase P) is composed of a catalytic RNA that is assisted by protein subunits. Holzmann et al. (2008) now provide evidence that the human mitochondrial RNase P is an entirely protein-based enzyme.

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing.

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.

Binding and cleavage of unstructured RNA by nuclear RNase P.

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.

Accumulation of noncoding RNA due to an RNase P defect in Saccharomyces cerevisiae.

Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 5' leader of pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in composition, as bacterial RNase P contains a catalytic RNA core and one protein subunit, while eukaryotic nuclear RNase P retains the catalytic RNA but has at least nine protein subunits. The additional eukaryotic protein subunits most likely provide additional functionality to RNase P, with one possibility being additional RNA recognition capabilities. To investigate the possible range of additional RNase P substrates in vivo, a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting that nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turn over pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively nonspecific capacity to bind and cleave mixed sequence RNAs, these data suggest that nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.

RNase P enzymes: divergent scaffolds for a conserved biological reaction.

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor-tRNAs (pre-tRNA) and is conserved in all domains of life. However, the composition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 proteins. Recently, a protein-only form of RNase P was discovered in mitochondria and chloroplasts of many higher eukaryotes. This proteinaceous RNase P (PRORP) represents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from Arabidopsis thaliana and speculate on the reasons for the replacement of catalytic RNA by a protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoprotein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the replacement of the RNA with a protein catalyst. The emergence of a protein-based RNase P more likely reflects the increasing complexity of the biological system, including difficulties in importation into organelles and vulnerability of organellar RNAs to cleavage.

Selection of RNA aptamers that bind HIV-1 LTR DNA duplexes: strand invaders.

RNA that can specifically bind to double-stranded DNA is of interest because it might be used as a means to regulate transcription of the target genes. To explore possible interactions between RNA and duplex DNA, we selected for RNA aptamers that can bind to the long terminal repeats (LTRs) of human immunodeficiency virus type 1 DNA. The selected aptamers were classified into four major groups based on the consensus sequences, which were found to locate in the non-stem regions of the predicted RNA secondary structures, consistent with roles in target binding. Analysis of the aptamer consensus sequences suggested that the conserved segments could form duplexes via Watson-Crick base-pairing with preferred sequences in one strand of the DNA, assuming the aptamer invaded the duplex. The aptamer binding sites on the LTR were experimentally determined to be located preferentially at these sites near the termini of double-stranded target DNA, despite selection schemes that were designed to minimize preferences for termini. The results presented here show that aptamer RNAs can be selected in vitro that strand-invade at preferred DNA duplex sequences to form stable complexes.

Spatial organization of genes as a component of regulated expression.

The DNA of living cells is highly compacted. Inherent in this spatial constraint is the need for cells to organize individual genetic loci so as to facilitate orderly retrieval of information. Complex genetic regulatory mechanisms are crucial to all organisms, and it is becoming increasingly evident that spatial organization of genes is one very important mode of regulation for many groups of genes. In eukaryotic nuclei, it appears not only that DNA is organized in three-dimensional space but also that this organization is dynamic and interactive with the transcriptional state of the genes. Spatial organization occurs throughout evolution and with genes transcribed by all classes of RNA polymerases in all eukaryotic nuclei, from yeast to human. There is an increasing body of work examining the ways in which this organization and consequent regulation are accomplished. In this review, we discuss the diverse strategies that cells use to preferentially localize various classes of genes.

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release.

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5' maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3' trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.

Genome-wide search for yeast RNase P substrates reveals role in maturation of intron-encoded box C/D small nucleolar RNAs.

Ribonuclease P (RNase P) is an essential endonuclease responsible for the 5'-end maturation of precursor tRNAs. Bacterial RNase P also processes precursor 4.5S RNA, tmRNA, 30S preribosomal RNA, and several reported protein-coding RNAs. Eukaryotic nuclear RNase P is far more complex than in the bacterial form, employing multiple essential protein subunits in addition to the catalytic RNA subunit. RNomic studies have shown that RNase P binds other RNAs in addition to tRNAs, but no non-tRNA substrates have previously been identified. Additional substrates were identified by using a multipronged approach in the budding yeast Saccharomyces cerevisiae. First, RNase P-dependant changes in RNA abundance were examined on whole-genome microarrays by using strains containing temperature sensitive (TS) mutations in two of the essential RNase P subunits, Pop1p and Rpr1r. Second, RNase P was rapidly affinity-purified, and copurified RNAs were identified by using a genome-wide microarray. Third, to identify RNAs that do not change abundance when RNase P is depleted but accumulate as larger precursors, >80 potential small RNA substrates were probed directly by Northern blot analysis with RNA from the RNase P TS mutants. Numerous potential substrates were identified, of which we characterized the box C/D intron-encoded small nucleolar RNAs (snoRNAs), because these both copurify with RNase P and accumulate larger forms in the RNase P temperature-sensitive mutants. It was previously known that two pathways existed for excising these snoRNAs, one using the pre-mRNA splicing path and the other that was independent of splicing. RNase P appears to participate in the splicing-independent path for the box C/D intron-encoded snoRNAs.

Broadening the mission of an RNA enzyme.

The "RNA World" hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA-protein complex plays diverse roles in processing RNA, but its best-understood reaction is pre-tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment.

RNase P: increased versatility through protein complexity?

Ribonuclease P (RNase P) is an essential enzyme that catalyzes the 5' endonucleolytic cleavage of precursor transfer RNAs (pretRNAs). It is found in all phylogenetic domains: bacteria, archaea and eukaryotes. The bacterial enzyme consists of a single, catalytic RNA subunit and one small protein, while the archaeal and eukaryotic enzymes have 4-10 proteins in addition to a similar RNA subunit. The bacterial RNA acts as a ribozyme at high salt in vitro; however the added protein optimizes kinetics and makes specific contacts with the pre-tRNA substrate. The bacterial protein subunit also appears to be required for the processing of non-tRNA substrates by broadening recognition tolerance. In addition, the immense increase in protein content in the eukaryotic enzymes suggests substantially enlarged capacity for recognition of additional substrates. Recently intron-encoded box C/D snoRNAs were shown to be likely substrates for RNase P, with several lines of evidence suggesting that the nuclear holoenzyme binds tightly to, and can cleave single-stranded RNA in a sequence dependent fashion. The possible involvement of RNase P in additional RNA processing or turnover pathways would be consistent with previous findings that RNase MRP, a variant of RNase P that has evolved to participate in ribosomal RNA processing, is also involved in turnover of specific messenger RNAs. Here, involvement of RNase P in multiple RNA processing pathways is discussed.

Prediction and verification of mouse tRNA gene families.

BACKGROUND: Transfer RNA (tRNA) gene predictions are complicated by challenges such as structural variation, limited sequence conservation and the presence of highly reiterated short interspersed sequences (SINEs) that originally derived from tRNA genes or tRNA-like transcription units. Annotation of "tRNA genes" in sequenced genomes generally have not been accompanied by experimental verification of the expression status of predicted sequences. RESULTS: To address this for mouse tRNA genes, we have employed two programs, tRNAScan-SE and ARAGORN, to predict the tRNA genes in the nuclear genome, resulting in diverse but overlapping predicted gene sets. From these, we removed known SINE repeats and sorted the genes into predicted families and single-copy genes. In particular, four families of intron-containing tRNA genes were predicted for the first time in mouse, with introns in positions and structures similar to the well characterized intron-containing tRNA genes in yeast. We verified the expression of the predicted tRNA genes by microarray analysis. We then confirmed the expression of appropriately sized RNA for the four intron-containing tRNA gene families, as well as the other 30 tRNA gene families creating an index of expression-verified mouse tRNAs. CONCLUSIONS: These confirmed tRNA genes represent all anticodons and all known mammalian tRNA structural groups, as well as a variety of predicted "rogue" tRNA genes within families with altered anticodon identities.

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture.

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

RNA affinity tags for the rapid purification and investigation of RNAs and RNA-protein complexes.

Isolation 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.

Spatial organization of transcription by RNA polymerase III.

RNA polymerase III (pol III) transcribes many essential, small, noncoding RNAs, including the 5S rRNAs and tRNAs. While most pol III-transcribed genes are found scattered throughout the linear chromosome maps or in multiple linear clusters, there is increasing evidence that many of these genes prefer to be spatially clustered, often at or near the nucleolus. This association could create an environment that fosters the coregulation of transcription by pol III with transcription of the large ribosomal RNA repeats by RNA polymerase I (pol I) within the nucleolus. Given the high number of pol III-transcribed genes in all eukaryotic genomes, the spatial organization of these genes is likely to affect a large portion of the other genes in a genome. In this Survey and Summary we analyze the reports regarding the spatial organization of pol III genes and address the potential influence of this organization on transcriptional regulation.

Effective expression of small interfering RNA in human cells.

In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a double-stranded RNA (dsRNA) corresponding to exon sequences in the mRNA (refs 1,2). The use of this "RNA interference" (RNAi) in mammalian studies had lagged well behind its utility in lower animals because uninterrupted RNA duplexes longer than 30 base pairs trigger generalized cellular responses through activation of dsRNA-dependent protein kinases. Recently it was demonstrated that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (approximately 20 base pairs) through liposome transfection. We have explored several strategies for expressing similar short interfering RNA (siRNA) duplexes within cells from recombinant DNA constructs, because this might allow long-term target-gene suppression in cells, and potentially in whole organisms. Effective suppression of target gene product levels is achieved by using a human U6 small nuclear RNA (snRNA) promoter to drive nuclear expression of a single RNA transcript. The siRNA-like parts of the transcript consists of a 19 base pair siRNA stem with the two strands joined by a tightly structured loop and a U1-4 3' overhang at the end of the antisense strand. The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.

Aggregation of Mod5 is affected by tRNA binding with implications for tRNA gene-mediated silencing.

Mod5 is a multifunctional protein that modifies a subset of tRNAs in the cytoplasm and is also required for an RNA-mediated form of transcriptional silencing. Previous in vivo studies have shown that the nuclear silencing function of Mod5 does not require that the causative tRNA gene encode a Mod5 substrate, although Mod5 is still required. However, previous data have not directly tested whether Mod5 can directly bind substrate and nonsubstrate RNAs. We herein demonstrate that Mod5 directly binds to both substrate and nonsubstrate RNAs, including a highly structured, non-tRNA sequence (5S-rRNA), consistent with previous in vivo data. Furthermore, we show that some RNAs drastically change the aggregation behavior of Mod5 with implications for tRNA gene-mediated silencing.

DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis.

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.