Discovery of a minimal form of RNase P in Pyrobaculum

L. B. Lai, P. P. Chan, A. E. Cozen, D. L. Bernick, J. W. Brown, V. Gopalan, T. M. Lowe
2010 Proceedings of the National Academy of Sciences of the United States of America  
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][1534][1535][1536][1537][1538][1539][1540]. A recent study concluded that N.
more » ... quitans 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. catalytic RNA | gene finding | RNA processing R Nase 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 (1-3). 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. Highthroughput 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 Author contributions: L.B.L. performed cloning, and designed and executed RNase P purification and pre-tRNA processing assays; P.P.C. performed promoter/sequencing analyses, identified Caldivirga and Vulcanisaeta RNase P RNAs, and created protein alignments; A.E.C. and D.L.B. grew Pyrobaculum cultures; A.E.C. purified total RNA, and performed northern analyses; D.L.B. designed and carried out RNA sequencing, and performed initial bioinformatic sequencing analyses; J.W.B. created secondary structure predictions for Pyrobaculum RNase P RNAs; V.G. guided biochemical studies; T.M.L. identified the Pyrobaculum RNase P RNAs, and analyzed the operonic context of RNase P proteins; L.B.L.
doi:10.1073/pnas.1013969107 pmid:21135215 pmcid:PMC3012483 fatcat:oezh2w7lk5b37ehe733w2x6h6i