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Proc Natl Acad Sci U S A. Dec 28, 2010; 107(52): 22493–22498.
Published online Dec 6, 2010. doi:  10.1073/pnas.1013969107
PMCID: PMC3012483
From the Cover
Biochemistry

Discovery of a minimal form of RNase P in Pyrobaculum

Abstract

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5′ leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533–1540]. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5′ tRNA leaders. The “missing” RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5′ pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5′ tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.

Keywords: catalytic RNA, gene finding, RNA processing

RNase P is best known for its role in removing the 5′ leaders of pre-tRNAs, an essential step in tRNA maturation. It also processes other RNAs in bacteria and eukaryotes, but these roles are less understood (13). RNase P typically functions as an RNA–protein complex, comprised of one conserved RNA and a varying number of protein subunits, depending on the domain of life: one in Bacteria, at least four in Archaea, and nine or more in the eukaryotic nucleus (4, 5). A precedent in which the RNA component is missing entirely is found in human and Arabidopsis organellar RNase P (6, 7), although a recent study suggests the possible coexistence of protein-only and RNA–protein-based RNase P complexes in human mitochondria (8).

The inability to identify RNase P in some organisms has sown doubts about whether it is a universal feature of life. Studies of the hyperthermophilic bacterium Aquifex aeolicus showed that it exhibits RNase P-like trimming of tRNAs (9, 10), yet a gene for the expected protein component is absent and the RNA has remained elusive (11), prompting speculation that it may have developed a unique solution for pre-tRNA processing (9). Perhaps most surprisingly, Söll and coworkers (12) demonstrated that the archaeal symbiont Nanoarchaeum equitans does not contain any identifiable RNase P genes or detectable RNase P activity, and it appears to lack tRNA leaders entirely. These findings leave RNase P conspicuously absent in just one other studied microbial species: Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon that has been refractory to prior biochemical (12) and computational (13, 14) identification efforts. Now, with the advent of new genome and RNA sequencing, augmented by improved computational search methods, we were able to uncover a unique form of RNase P in multiple Pyrobaculum species and related genera.

Results and Discussion

Pre-tRNAs in Pyrobaculum Have 5′ Leaders.

We first obtained evidence for RNase P activity in Pyrobaculum using comparative genomics and RNA sequencing. The genomes of four Pyrobaculum species [Pyrobaculum arsenaticum, Pyrobaculum calidifontis, Pyrobaculum islandicum, and Thermoproteus neutrophilus (to be reclassified as a Pyrobaculum species)] were recently sequenced in collaboration with the Joint Genome Institute, providing extensive comparative information. As in P. aerophilum, the RNase P RNA genes could not be identified in these genomes using existing computational methods (13, 14) (see Materials and Methods). However, alignment of orthologous tRNA loci and upstream promoter regions from all these Pyrobaculum species provided compelling evolutionary evidence for pre-tRNA leaders, thus hinting at a requirement for RNase P. If no RNase P activity is present to remove 5′ leaders, then one should expect very little or no variation in the distance between the TATA sequence and the 5′ end of the mature tRNA gene, especially among orthologs. In fact, we counted more than 12 tRNA ortholog groups where the distance between the promoter and tRNA gene varies by at least two nucleotides among species (Fig. S1). Next, we examined native transcripts from tRNA loci for four of these species to confirm the computational observations. High-throughput RNA sequencing reads of small RNAs identified many tRNA transcripts with 5′ leaders: 15 in P. aerophilum, 17 in P. arsenaticum, 18 in P. calidifontis, and 21 in P. islandicum have 1- to 6-nt leaders (Table 1 shows counts by leader length). All of the sequenced pre-tRNAs with 5′ leaders were also found in their mature form. These data strongly suggest that Pyrobaculum has some form of pre-tRNA 5′-processing activity.

Table 1.
tRNA genes found to have a transcribed 5′ leader by high-throughput RNA sequencing

Pyrobaculum aerophilum Cell Extract Processes 5′ Leader from pre-tRNA.

Because RNase P activity was previously not found in a P. aerophilum crude extract (12), we opted to assay this activity after chromatographic fractionation. When a cell-free extract of P. aerophilum was subjected to successive weak cation (CM)- and anion (DEAE)-exchange matrices, a peak of 5′-processing activity (Fig. S2) was observed with a P. aerophilum pre-tRNAPhe (Fig. 1A) as the substrate. The products generated were identical in size to those obtained with in vitro reconstituted Escherichia coli RNase P (15) when pre-tRNAPhe containing uridine at the -1 position (U-1) was used (Fig. 1B); cleavage took place as expected between nucleotides -1 and +1 (Fig. S3 provides additional data characterizing cleavage-site selection). Another hallmark of RNase P-mediated processing is the presence of a 5′ phosphate in the mature tRNA product (15). TLC of mature tRNAPhe generated by P. aerophilum RNase P and subsequently digested with RNase T2 showed the presence of a 5′ phosphate on G+1 (pGp in Fig. 1C), as observed with E. coli RNase P. Collectively, these results establish that the partially purified pre-tRNA 5′-processing activity in P. aerophilum is indeed RNase P.

Fig. 1.
The pre-tRNA 5′-processing activity from P. aerophilum (Pae) cell extract has all the cleavage properties of RNase P. (A) A P. aerophilum pre-tRNAPhe with an 18-nt leader used for RNase P assays; pre-tRNAPhe (G-1) and pre-tRNAPhe (U-1) differ ...

Evidence for Three Out of Four Known Archaeal RNase P Proteins in Pyrobaculum.

All known archaeal RNase P complexes require both RNA and protein components, so we tested P. aerophilum RNase P for these constituents. Treatment with proteinase K, which degrades proteins, eliminated the P. aerophilum RNase P activity (Fig. 2A), raising the question of the protein component identities. Of the four established archaeal RNase P proteins (Rpp29, Rpp30, Pop5, Rpp21), Pfam (16) and other sequence profile searches identified Pyrobaculum orthologs for Rpp29 (first noted by Hartmann and Hartmann; ref. 17), gave weak support for Rpp30 candidates, but had no predictions for Pop5 or Rpp21 orthologs. Koonin et al. (18) previously noted that the Rpp30 and Pop5 genes are sometimes adjacent to each other and are often in the same operon as the gene for a predicted exosome protein (DUF54/COG1325) (18). In Pyrobaculum species, this exosome gene clearly identifies a well-conserved operon that contains both the candidate Rpp30 genes and plausible Pop5 homologs (Table S1). Alignment of the Pyrobaculum candidates (PAE1830 for Rpp30; PAE1829 for Pop5) with the known archaeal protein homologs reveals potential homology: The Pyrobaculum candidates retain most of the highly conserved residues, but have lost some conserved segments, making the proteins 10–25% shorter than known archaeal family members. Alignment of the predicted Rpp29 ortholog (PAE1777) to archaeal Rpp29 proteins provides similar positive support. Further operon and sequence analyses, including a highly sensitive structure-based protein search, failed to identify the Rpp21 ortholog in any Pyrobaculum genome. These results suggest that only three of the four known archaeal RNase P proteins exist in Pyrobaculum.

Fig. 2.
RNase P activity from P. aerophilum (Pae) cell extract requires both protein and RNA subunits. (A) Treatment with proteinase K (PK) eliminated Pae RNase P activity. Pae RNase P was preincubated with PK (lane 3) or without PK (lane 4) before assaying for ...

Discovery and in Vitro Activity of the Minimized Pyrobaculum RNase P RNA.

Given these apparent changes in the RNase P proteins, it was still unclear how the missing Pyrobaculum RNA might have changed to elude prior detection. Therefore, we made no assumptions about the RNA’s primary sequence length or secondary structure features. Using the University of California Santa Cruz Archaeal Genome Browser (19) (archaea.ucsc.edu), we aligned the four new Pyrobaculum genomes with P. aerophilum, allowing us to focus on the most highly conserved regions common to all five species. After removing known RNA genes (rRNAs, tRNAs, C/D box sRNAs, signal-recognition particle RNA), only a few regions were conserved > 90%, forming an enriched group to study. In one of these conserved regions, we detected a very weak partial hit to archaeal RNase P RNA using the Infernal RNA search program (20), but we also observed properties expected in good candidates: high (73–77%) guanine/cytosine content typical for a hyperthermophilic structural RNA, a strong transcription factor B recognition element (BRE)-TATA promoter sequence upstream, and extremely high conservation (Fig. 3). Although the encoded RNA is much shorter than any identified nonorganellar RNase P RNAs (21), the Pyrobaculum candidates can be folded into an RNase P-like consensus secondary structure (Fig. 4A and Fig. S4) with one surprising difference. All known bacterial and archaeal RNase P RNAs consist of both a substrate-specificity (S) domain that aids substrate recognition, and a catalytic (C) domain essential for phosphodiester cleavage (22, 23). The Pyrobaculum RNase P RNA candidates have lost most of their S domain, but retain an intact C domain that includes all 11 universally conserved nucleotides (24) (Fig. 4A and Fig. S4).

Fig. 3.
P. aerophilum RNase P RNA displayed on the Archaeal Genome Browser (19) and alignment of the RNase P RNA sequences from five Pyrobaculum species. (A) The red segment located between protein-coding genes PAE0934 and PAE0936 corresponds to the P. aerophilum ...
Fig. 4.
Predicted secondary structure, native expression, and in vitro activity of P. aerophilum (Pae) and Caldivirga maquilingensis (Cma) RNase P RNAs (RPRs). (A) The Pae RPR structure shared by four other Pyrobaculum spp., with unlabeled nucleotides identical ...

To verify the candidate P. aerophilum RNase P RNA, we first confirmed expression by northern analysis of total RNA (Fig. 4B). Second, to assess association of this RNA with the partially purified, native P. aerophilum RNase P holoenzyme, we designed PaeRPR-L15, an antisense 14-mer RNA oligonucleotide which we expected would invade an essential loop region in the RNA moiety (nucleotides 157–170; Fig. 4A) and thereby interfere with activity. As shown for bacterial RNase P (25), we observed a progressive decrease in native P. aerophilum RNase P activity when the enzyme was preincubated with increasing concentrations of this oligonucleotide (Fig. 2B) and that the extent of this inhibition was more pronounced compared to that observed with a nonspecific oligonucleotide (see SI Text). We did not expect complete inhibition because PaeRPR-L15’s access to the RNA moiety of P. aerophilum RNase P could be hampered by enveloping protein subunits. Such a premise is consistent with our findings that P. aerophilum RNase P could not be inactivated with micrococcal nuclease, for which there is a precedent in another archaeal hyperthermophile (26). Last, but most importantly, an in vitro transcript of the P. aerophilum candidate RNase P RNA was able to process pre-tRNAPhe (Fig. 4C and Fig. S5), confirming it is indeed the missing RNase P ribozyme. As a negative control, deletion of the universally conserved, bulged U51 (Fig. 4A) in the same transcript eliminated the RNA-alone activity (Fig. 4C, lane 2); this bulged U in bacterial RNase P RNA appears to be required for maintaining a unique geometry essential for substrate positioning and binding of catalytically important Mg2+ ions (27). As with the partially purified native enzyme, preincubation of the in vitro RNase P RNA transcript with PaeRPR-L15 drastically decreased its activity as expected.

Phylogenetic Distribution of the Minimized Form of RNase P RNA.

Equipped with a broader view of RNase P RNA, we surveyed all 88 currently available, complete archaeal genomes to determine which have traditional, Pyrobaculum-like, or possibly other missing and/or undetectable versions. To search for traditional RNase P RNA genes, we used the Rfam archaeal RNase P covariance model (14) and the rule-based method by Li and Altman (13). Although slower, the covariance model was much more sensitive, finding matches in every species except N. equitans, Pyrobaculum spp., and unexpectedly, Caldivirga maquilingensis and Vulcanisaeta distributa, two crenarchaea in the same family (Thermoproteaceae) as Pyrobaculum (Table S2). By employing a covariance model based on the unusual Pyrobaculum RNAs, we uncovered a similarly shortened form of RNase P RNA in C. maquilingensis and V. distributa (Fig. 4A, Fig. S4, and Table S2), now accounting for an RNase P RNA gene in all complete archaeal genomes except N. equitans (12). We established expression of the C. maquilingensis candidate RNase P RNA in vivo (Fig. 4B) and that an in vitro transcript of this RNA can process pre-tRNAPhe (Fig. 4C and Fig. S5). Like its Pyrobaculum counterpart, deletion of the bulged U63 together with C62 and C64 in the C. maquilingensis transcript eliminated the RNA-alone activity (Fig. 4C).

Both of these RNAs display all the hallmarks of other RNase P RNAs: They cleave at the expected site (Fig. S3B), they generate 5′-phosphate and 3′-hydroxyl termini (Fig. 1C and Fig. S5C), and they are capable of cleaving pre-tRNAPhe with a short and long leader (Fig. S5B). Taken together, these data suggest that our Pyrobaculum-based covariance search model is complementary to the existing Rfam RNase P model and should facilitate detection of other shortened forms of RNase P (dubbed “type T” for the phylogenetic family Thermoproteaceae).

A search for the RNase P proteins in Caldivirga and Vulcanisaeta revealed likely homologs for Pop5, Rpp30, and Rpp29 (see SI Text), but, as in Pyrobaculum, no apparent ortholog of Rpp21. Because Pyrobaculum, Caldivirga, and Vulcanisaeta are closely related genera, it is most parsimonious to assume that the type T RNase P RNA was a feature of the common ancestor of this family. Outside of Thermoproteaceae, the next most closely related species with a sequenced genome is Thermofilum pendens, which seems to possess a typical RNase P RNA (14) with a traditional S domain and all four traditional archaeal RNase P proteins (Tables S1 and S2). Thus, the conspicuous absence of Rpp21 in the Thermoproteaceae may reflect fewer cognate RNase P proteins for the smaller type T RNA, or that Rpp21 has diverged too extensively to be detected by sequence similarity searches. Reconstitution studies have revealed that the archaeal RNase P proteins function as two binary complexes: Pop5•Rpp30 and Rpp21•Rpp29 (23). Footprinting studies indicate that Pop5•Rpp30 interacts with the C domain, and Rpp21•Rpp29 interacts with the S domain of archaeal RNase P RNA (23, 28). Thus, the loss or radical change of Rpp21 would be consistent with loss of the traditional S domain in type T RNase P RNAs.

Search for RNase P RNA in Aquifex and Related Species.

With the identification of the smaller RNase P RNA in Pyrobaculum and related archaeal species, we decided to apply existing and our newly developed covariance search models to four bacterial species in which RNase P RNA appears to be absent. These species include the well-studied Aquifex aeolicus, as well as three other species also in the Aquificaceae family (Hydrogenivirga sp. 128-5-R1-1, Hydrogenobacter thermophilus, and Hydrogenobaculum sp. Y04AAS1). We searched these genomes using all existing bacterial, archaeal, and eukaryotic RNase P RNA models in Rfam (14), as well as our Pyrobaculum-specific model, producing no good candidates. Because there are significant structural differences between bacterial and archaeal RNase P RNAs (4), we also created a model based on the RNase P RNAs from the closest related bacterial species that have known RNase P RNAs and artificially removed the specificity domain. The shortened bacterial search model produced no good candidates in any of the four genomes.

Conclusions

The reduced size of the type T form of RNase P RNA may correlate with unique features of this group of organisms. Pyrobaculum and Vulcanisaeta species are unusual for the large number of tRNAs with more than one intron, many at “noncanonical” positions (29, 30). Caldivirga does not have as many atypical tRNA introns, but does contain a number of trans-spliced split tRNAs (31), a rare trait shared only with N. equitans (32). It is unclear if changes in the pre-tRNA structures are related to the altered substrate recognition domain, but a link would not be surprising.

Our discovery of the drastically smaller type T RNase P emphasizes the striking and surprising plasticity in the subunit composition of an essential and ubiquitous enzyme. This flexibility may be an adaptive trait related to the variability in the RNAs that are substrates for RNase P. In bacteria, these assorted substrates include 4.5S, tmRNA, viral RNAs, riboswitches, and some mRNAs (2). In Thermoproteaceae, type T RNase P may play a similarly expanded role in small RNA processing.

Collectively, our findings highlight the rich evolutionary story of RNase P and offer opportunities for structural, comparative, and functional studies. As the cost of genome and transcriptome sequencing continues to decrease, we expect the combination of comparative genomics and improved RNA search models will continue to reveal exceptional cases advancing RNA biology.

Materials and Methods

Archaeal RNase P RNA Sequence Search.

Infernal v1.0 (20) was used to search for RNase P RNA candidates in archaeal genomes. The program was initially run in the global search mode using the Rfam (14) archaeal RNase P RNA covariance model (RF00373). All hits with a score > 0 bits were manually examined. Local search mode was also employed, which provided better sensitivity but decreased selectivity. Using local search mode with a threshold of 0 bits (necessary to initially detect the Pyrobaculum RNase P RNA with the RF00373 model) was only feasible when searching a small set of short candidate regions (i.e., not entire genomes) because low-scoring partial hits required close manual inspection and further structural analyses.

The rule-based program by Li and Altman (13) was used to search for RNase P RNA candidates in all available archaeal genomes retrieved from ftp://ftp.ncbi.nih.gov/genomes/Bacteria/. The genome of Vulcanisaeta distributa was downloaded from the publicly accessible Integrated Microbial Genomes with the Genomic Encyclopedia of Bacteria and Archaea Genomes Web site (http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=TaxonDetail&taxon_oid=2502790013). Because hits were not found in a number of genomes, Infernal (global search mode) was applied using the Rfam RF00373 model. A universal primary sequence pattern for RNase P RNA, modified from the original Li and Altman pattern, was used to prescreen candidates that were then analyzed with Infernal. For archaeal genomes producing no hits using this fast prescreening method, whole genome searches were conducted with Infernal alone (greatly slowing search speed, but improving search sensitivity). The Pyrobaculum RNase P RNA covariance model was built with Infernal using the five Pyrobaculum RNA sequences and a manually predicted secondary structure (Fig. 4A).

RNase P Purification.

P. aerophilum cells were grown as described in SI Materials and Methods. One gram of cells was resuspended in 5 mL cold extraction buffer (EB; 20 mM Tris•HCl, pH 8 at 25 °C and 5 mM MgCl2) containing 50 mM NaCl, 10 mM DTT, and 1 mM PMSF. Cell-free extract was generated by sonication and clarified by centrifugation at 14,000  × g and 4 °C for 30 min. The crude extract was then loaded on a 1-mL HiTrap CM FF Sepharose column (GE Healthcare). EB supplemented with various NaCl concentrations, 10% (vol/vol) glycerol, 2 mM DTT, and 0.2 mM PMSF was used in all the following steps. With an FPLC apparatus, fractions were eluted using a 50–2000 mM NaCl gradient and subsequently assayed for RNase P activity (33). The active CM fractions were pooled, dialyzed, and loaded on a 1-mL HiTrap DEAE FF Sepharose column (GE Healthcare). The activity was eluted with three sequential NaCl gradients: 50–400, 400–1,000, and 1,000–1,250 mM. The peak of RNase P activity eluted at ~700 mM NaCl.

RNase P Assay.

The P. aerophilum pre-tRNAPhe was in vitro transcribed and radioactively labeled either internally using [α-32P]-GTP during transcription or at the 5′ end using T4 polynucleotide kinase (New England Biolabs, NEB) and [γ-32P]-ATP after dephosphorylation of the transcript by calf intestinal alkaline phosphatase (NEB). RNase P activity was assayed at 55 °C in 50 mM Tris•HCl (pH 7.5 at 25 °C), 400 mM NH4OAc, 10 mM Mg(OAc)2, 5 mM DTT, 0.1 unit/μL RiboLock (Fermentas), 100 nM unlabeled pre-tRNAPhe, and trace amounts of labeled pre-tRNAPhe (~1 nM). Activity of the in vitro transcribed P. aerophilum and C. maquilingensis RNase P RNA was assayed, without a prefolding incubation, at 55 °C for 20 h in 50 mM Tris•HCl (pH 7.5 at 25 °C), 1.5 M NH4OAc, trace amounts (~1 nM) of labeled pre-tRNAPhe, and Mg(OAc)2 at 75 and 100 mM, respectively. The cleavage products were then separated on 12% (wt/vol) polyacrylamide/7 M urea gels (or 15% for the 4-nt leadered pre-tRNAPhe) and radiographed on a PhosphorImager.

TLC.

Subsequent to cleavage of internally labeled pre-tRNAPhe by P. aerophilum and E. coli RNase P or P. aerophilum, C. maquilingensis and E. coli RNase P RNA, the mature tRNAPhe products were purified using an 8% (wt/vol) polyacrylamide/7 M urea gel. Uncleaved internally labeled pre-tRNAPhe was used as a negative marker and uncleaved 5′ end-labeled pre-tRNAPhe (with the 5′ monophosphorylated G being the only labeled residue) as the positive marker of pGp. These RNAs were then completely digested with 12 units of RNase T2 (Gibco BRL) at 50 °C for 1 h in 20 mM NaOAc (pH 5.2 at 25 °C), 1 mM EDTA, and 0.4 μg/μL yeast tRNAs. Each sample was spotted on a TLC PEI cellulose F plate (EMD Chemicals), separated with 0.5 M potassium phosphate (pH 6.3):methanol in an 80[ratio]20 ratio, and radiographed with a PhosphorImager.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank J. Murphy [University of California, Santa Cruz (UCSC)] for her contribution of Pyrobaculum cell material, L. Lui (UCSC) for her contribution of Caldivirga cell material for northern analysis, and K. Karplus (UCSC) for his Rpp21 structural protein search and expert advice on protein alignment interpretation. We are grateful to members of the Joint Genome Institute for making 454 sequencing possible (P. Richardson and J. Bristow for providing resources, and E. Lindquist and N. Zvenigorodsky for sample preparation and analysis). We thank J. Jackman [Ohio State University (OSU)] for advice and reagents for the TLC analysis, and E. J. Behrman (OSU) for helpful discussions. We are indebted to S. Eddy (Howard Hughes Medical Institute Janelia Farm) for insightful comments that helped improve the manuscript. This work was supported by National Science Foundation Grants MCB0238233 and MCB0843543 (to V.G.) and EF-0827055 (to T.L.), and National Institutes of Health GM067807 (to Mark P. Foster and V.G.) and HG004002-02 subaward (to T.L.).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

See Commentary on page 22371.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1013969107/-/DCSupplemental.

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