<i>Drosophila RPR</i> is embedded in an intron and ubiquitously expressed.

<p>A. The <i>RPR</i> gene (pink) in <i>D. melanogaster</i> is present in the last intron of <i>ATPsynC</i>/<i>CG1746</i>. B. This arrangement is conserved in <i>D. pseudoobscura</i> (and other members of the genus<i>).</i> The exons of <i>ATPsynC</i> (orange peaks) are highly conserved between these species, as is the region within the intron that contains <i>RPR</i> (pink). The preceding intron is not conserved. Untranslated regions of <i>ATPsynC</i> are shown in grey. Peaks showing 75% or greater conservation are colored. C. Analysis of polyA-selected RNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Graveley1" target="_blank">[52]</a> from <i>D. pseudoobscura</i> and <i>D. virilis,</i> and of total RNA from different developmental stages of <i>D. melanogaster</i> show that the region corresponding to <i>RPR</i> is expressed at higher levels than the preceding intron <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Graveley1" target="_blank">[52]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Roy1" target="_blank">[53]</a>. Presence of RPR in polyA⁺ RNA is likely due to carryover (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#s4" target="_blank">Materials and Methods</a>). D. ChIP on chip data (<i>D. melanogaster</i> embryos) showing binding sites of pol II <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Li2" target="_blank">[63]</a> and transcription factor IIB (TF-IIB) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-MacArthur1" target="_blank">[64]</a> in the 5′ region of <i>ATPsynC/CG1746</i>. E, embryonic stage in hours after egg laying; L, larval instar; WPP, white pre-pupae; F, female; M, male.</p

Insects and crustaceans have <i>RPR</i> genes embedded in <i>pol II</i> recipient genes, while other animals have independent <i>pol III</i> genes.

<p>The location of <i>RPR</i> and the neighboring genes in representative species of insects, crustaceans, and other animals are shown. In insects and crustaceans (light grey), <i>RPR</i> genes lack pol III signals and are in an intron (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893.s002" target="_blank">S2 Fig.</a>). The <i>P. humanus</i> gene lacks pol III signals and is currently annotated between two genes. Each recipient gene is color-coded (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen-1004893-g005" target="_blank">Fig. 5</a>; homologous genes have the same color). In other sub-phyla of Arthropoda (Myriapoda and Chelicerata) and other phyla (Deuterostomia and Porifera) (dark grey), <i>RPR</i> is an independent pol III-regulated gene (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893.s002" target="_blank">S2 Fig.</a>). <i>RPRs</i> without pol III signals, pink; <i>RPRs</i> with pol III signals, blue; proximal sequence element (PSE), blue star; TATA box 21–27 nucleotides upstream of RPR, green oval; 3′ poly-T stretch of 4–5 nucleotides, red triangle. Wavy lines indicate regions where either poor sequence quality or weak homology prevents accurate prediction of the exons. Scale bar is 1 kb.</p

RPR is processed from a recipient intron.

<p>Various <i>RFP</i> reporter genes harboring the RPR<i>-</i>coding <i>D. virilis</i> intron were expressed in <i>D. melanogaster</i> S2 cultured cells to define sequences required for biogenesis of RPR. A. Schematic showing the reporter genes tested. B. <i>RFP</i> pre-mRNA and mRNA were analyzed by RT-PCR (using the primers FP.RT and RP.RT indicated in A). The presence of the protein was determined by fluorescent microscopy. C. RPR was detected by northern analysis. The antisense <i>D. virilis</i> RNA probe also detected the native <i>D. melanogaster</i> RPR because of sequence conservation. <i>Controls used</i>: GFP, expressed from a co-transfected plasmid to serve as a control for transfection efficiency; <i>Oda</i> (Ornithine decarboxylase antizyme) is a housekeeping transcript used to normalize input RNA for the RT-PCR experiment; U6 RNA was used as loading control the northern analysis.</p

Model for the evolutionary history of <i>RPR</i>.

<p>A. An ancestral <i>RPR</i> gene is thought to have undergone gene duplication and one of the daughter genes assumed the new functions of MRP RNA (neofunctionalization). <i>MRP RNA</i> is transcribed by pol III in all animals, as is <i>RPR</i> in animals previously characterized. We found that the <i>RPR</i> gene in crustaceans and insects has undergone another genetic event that inserted it, devoid of pol III signals, into a pol II-transcribed gene. B. A cladogram showing arthropod evolution (based on, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Regier1" target="_blank">[26]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Oakley1" target="_blank">[27]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Giribet1" target="_blank">[65]</a>). In Hexapods and true crustaceans (Vericrustacea) (light grey), <i>RPR</i> is embedded in a pol II-regulated gene. In contrast, in Myriapoda and Chelicerata, <i>RPR</i> is a pol III gene (dark grey). The <i>RPR</i> genes in Remipedia and Oligostraca have not been characterized due to lack of genomic sequences (unshaded). The arrow indicates a node that connects branches where <i>RPR</i> is found in a recipient gene. These groups are thought to have diverged 500 million years ago <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Misof1" target="_blank">[18]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Glenner1" target="_blank">[66]</a>. We predict that Remipedia <i>RPR</i> is also embedded in a recipient gene similar to the sister group Hexapoda. An analysis of <i>RPR</i> in Oligostraca will enable us to determine if embedding of <i>RPR</i> occurred earlier in arthropod evolution in an ancestor of all pancrustaceans.</p

Transcriptional Control of an Essential Ribozyme in <i>Drosophila</i> Reveals an Ancient Evolutionary Divide in Animals

<div><p>Ribonuclease P (RNase P) is an essential enzyme required for 5′-maturation of tRNA. While an RNA-free, protein-based form of RNase P exists in eukaryotes, the ribonucleoprotein (RNP) form is found in all domains of life. The catalytic component of the RNP is an RNA known as RNase P RNA (RPR). Eukaryotic <i>RPR</i> genes are typically transcribed by RNA polymerase III (pol III). Here we showed that the <i>RPR</i> gene in <i>Drosophila</i>, which is annotated in the intron of a pol II-transcribed protein-coding gene, lacks signals for transcription by pol III. Using reporter gene constructs that include the RPR-coding intron from <i>Drosophila</i>, we found that the intron contains all the sequences necessary for production of mature RPR but is dependent on the promoter of the recipient gene for expression. We also demonstrated that the intron-coded RPR copurifies with RNase P and is required for its activity. Analysis of <i>RPR</i> genes in various animal genomes revealed a striking divide in the animal kingdom that separates insects and crustaceans into a single group in which <i>RPR</i> genes lack signals for independent transcription and are embedded in different protein-coding genes. Our findings provide evidence for a genetic event that occurred approximately 500 million years ago in the arthropod lineage, which switched the control of the transcription of <i>RPR</i> from pol III to pol II.</p></div

Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29

We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg²⁺-dependent cleavage of the 5′ leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5–RPP30 and RPP21–RPP29. Nuclear magnetic resonance studies of the <i>Pyrococcus furiosus</i> RPP21 and RPP29 proteins in their free and complexed states provided evidence of significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, and pH values, in buffers with varying ionization potentials, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (Δ<i>C</i>_<i>p</i>), nearly twice that predicted from surface accessibility calculations, a strong salt dependence for the interaction, and proton release at neutral pH, but a small net contribution from these to the excess Δ<i>C</i>_<i>p</i>. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess Δ<i>C</i>_<i>p</i>. These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies

<i>RPR</i> genes lacking <i>pol III</i> signals are only present in the arthropod clade that includes insects and crustaceans.

<p>Phylogenetic relationship of animals showing two groups, those with <i>RPR</i> genes lacking pol III signals (light grey) and others with typical motifs found in type 3 pol III genes (dark grey). (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893.s002" target="_blank">S2 Fig.</a> for sequence motifs). The divide occurs in Arthropoda—species of Insecta and Vericrustacea (true crustaceans, including branchiopods and copepods) have <i>RPR</i> genes that lack pol III signals, whereas species of Myriapoda and Chelicerata have <i>RPR</i> genes with typical pol III signals. The <i>RPR</i> genes are associated with a variety of different recipient genes, indicated by different colored bars and named in the key (the same scheme is used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen-1004893-g004" target="_blank">Fig. 4</a>). In crustaceans (cyan), where there are multiple <i>RPR</i> genes in a single species, none is inserted in the ortholog of a gene identified as a recipient gene in insects.</p

<i>D. melanogaster</i> RPR co-purifies with RNase P and is required for its activity.

<p>A. RNase P activity was partially purified from <i>D. melanogaster</i> S2 cells using sequential DEAE- and SP-Sepharose (above). Pre-tRNA processing assays established that the peak of activity eluted in 300–500 mM NaCl (fractions 3–5). RNA isolated from all fractions was subjected to RT-PCR using RPR-specific primers. Amplicons corresponding to the expected RPR size were detected in fractions 3–5 that showed maximal RNase P activity. B. Thin-layer chromatographic analysis of RNase T2-cleaved tRNA^Gly containing a 5′-pGp; the tRNA^Gly was first generated from cleavage of internally [α-³²P]-GTP-labeled pre-tRNA^Gly by <i>in vitro</i> reconstituted <i>E. coli</i> RNase P or partially-purified <i>D. melanogaster</i> RNase P (lanes 1 and 4, respectively). The negative control (lane 3) shows RNase T2-cleaved internally labeled pre-tRNA^Gly that lacks a 5′-pGp, and the positive control (lane 2) shows RNase T2-cleaved 5′-labeled pre-tRNA^Gly that has a 5′-pGp. C. The predicted secondary structure of <i>D. melanogaster</i> RPR contains universally-conserved and functionally-important nucleotides (indicated by black circles). An antisense RNA oligonucleotide (red line; α-RPR-j7/2, complementary to a predicted single-stranded region between paired regions P7 and P2) was designed to inhibit RNase P activity. D. Partially-purified RNase P was inactivated with increasing concentrations of α-RPR-j7/2, but not with a scrambled oligo (sc-RPR-j7/2) that has the same nucleotide composition as α-RPR-j7/2. NC, negative control with no enzyme added; PC, positive control with <i>in vitro</i> reconstituted <i>E. coli</i> RNase P; IP, input; FT, flow-through.</p

Frequencies of cleavage-site selection by <i>At</i>PRORP1.

<p>Histograms summarizing cleavage-site selection frequencies (in %) during <i>At</i>PRORP1-mediated cleavage of pSu1 "-1" (A) and pATSer (B) variants. Mean and standard deviation values were calculated using data from at least three independent experiments.</p

<i>At</i>PRORP1-mediated cleavage of pre-tRNA^SerSu1 (pSu1).

<p>Representative gel showing <i>At</i>PRORP1-mediated cleavage of pre-tRNA^SerSu1 (pSu1) substrates with and without the 3' CCA. Lanes 1 to 8 represent negative controls (absence of <i>At</i>PRORP1), and M (size marker, lane 9) indicates cleavage of pATSerUG by <i>Eco</i> RPR. Note that this cleavage generates a 5' cleavage fragment (5' CL Frags) one nucleotide longer compared to that generated during cleavage of pSu1. Lanes 10 and 14 pSu1(-1A), lanes 11 and 15 pSu1(-1C), lanes 12 and 16 pSu1(-1G), and lanes 13 and 17 pSu1(-1U). The final concentration of <i>At</i>PRORP1 was 0.37 μM and the reactions were performed at 37°C for 30 s in the presence of 10 mM Mg²⁺ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160246#sec002" target="_blank">Materials and Methods</a>).</p