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Piece by piece: Building a ribozyme

Open AccessPublished:January 17, 2020DOI:https://doi.org/10.1074/jbc.REV119.009929
      The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient biological assemblies that were already present in the last universal common ancestor of all life. The large subunit rRNA in the ribosome and the RNA subunit of RNase P are the ribozyme components required for catalysis. Here, we explore the idea that these two large ribozymes may have begun their evolutionary odyssey as an assemblage of RNA “fragments” smaller than the contemporary full-length versions and that they transitioned through distinct stages along a pathway that may also be relevant for the evolution of other non-coding RNAs.

      Introduction

      The ribosome and RNase P stand out among all cellular ribonucleoprotein (RNP)
      The abbreviations used are: RNP
      ribonucleoprotein
      LSU
      large subunit
      LUCA
      last universal common ancestor
      mitoribosome
      mitochondrial ribosome
      mtDNA
      mitochondrial DNA
      ncRNA
      non-coding RNA
      nt
      nucleotide(s)
      PTC
      peptidyl transferase center
      RP
      ribosomal protein
      RPR
      RNase P RNA
      SRP
      signal recognition particle
      SSU
      small subunit
      tmRNA
      transfer-messenger RNA
      ITS
      internal transcribed spacer.
      complexes. These two RNPs, which play central roles in translation and tRNA processing, respectively, are ancient biological entities that were already present in the last universal common ancestor (LUCA) of all life (
      • Evans D.
      • Marquez S.M.
      • Pace N.R.
      RNase P: interface of the RNA and protein worlds.
      ,
      • Gopalan V.
      Uniformity amid diversity in RNase P.
      ,
      • Ellis J.C.
      • Brown J.W.
      The RNase P family.
      ,
      • Hsiao C.
      • Mohan S.
      • Kalahar B.K.
      • Williams L.D.
      Peeling the onion: ribosomes are ancient molecular fossils.
      ,
      • Fox G.E.
      Origin and evolution of the ribosome.
      ,
      • Daniels C.J.
      • Lai L.B.
      • Chen T.-H.
      • Gopalan V.
      Both kinds of RNase P in all domains of life: surprises galore.
      ), emerging prior to the divergence of living organisms into the primary domains: Archaea, Bacteria, and Eucarya (
      • Woese C.R.
      • Fox G.E.
      Phylogenetic structure of the prokaryotic domain: the primary kingdoms.
      ,
      • Woese C.R.
      • Kandler O.
      • Wheelis M.L.
      Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya.
      ). In eukaryotes, they function in the nucleus/cytoplasm and in protein-synthesizing organelles (mitochondria and plastids). This early emergence and long evolutionary history emphasize the fundamental and essential biological roles played by these two pervasive RNPs. Notably, peptide bond synthesis and phosphodiester bond cleavage, respectively, are catalyzed by the large subunit ribosomal RNA (rRNA) and the RNA subunit of RNase P (RPR) in the corresponding RNPs. The recognition of RNA-mediated catalysis in the self-splicing group I intron and RNase P, as well as the ribosome, constituted a true paradigm shift in our understanding of the cellular catalytic repertoire (
      • Guerrier-Takada C.
      • Gardiner K.
      • Marsh T.
      • Pace N.
      • Altman S.
      The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme.
      ,
      • Cech T.R.
      The ribosome is a ribozyme.
      ,
      • Steitz T.A.
      • Moore P.B.
      RNA, the first macromolecular catalyst: the ribosome is a ribozyme.
      ).
      An intriguing unsolved puzzle relates to the evolution of the two large ribozymes at the heart of the ribosome and RNase P. These catalytic RNAs may represent relics of a primordial RNA world (
      • Robertson M.P.
      • Joyce G.F.
      The origins of the RNA world.
      ), the implication being that these RNAs were functional before their transition into contemporary, multisubunit RNP forms. Indeed, Crick (
      • Crick F.H.C.
      The origin of the genetic code.
      ) stated explicitly that “the primitive ribosome could have been made entirely of RNA,” an early view also championed by Woese (
      • Woese C.R.
      The emergence of genetic organization.
      ,
      • Woese C.R.
      Just So stories and Rube Goldberg machines: speculations on the origin of the protein synthetic machinery.
      ). In this review, we explore the premise that large ribozymes may have begun their evolutionary odyssey as an assemblage of RNA “fragments” smaller than the existing, full-length versions, a likely first step for the domain accretion that followed. Trajectories of extant macromolecules, deduced from the footprints of evolution, have loose ends not unlike any complicated tapestry. Notwithstanding these potential gaps, the extant diversity of biogenesis routes and structures of rRNA and RPR has shaped our narrative on the evolution of these two ribozymes.

      Evolution of large ribozymes may have begun with smaller, isolated fragments

      rRNA and RPR are long, covalently continuous RNA molecules whose secondary structures are characterized by an interspersed pattern of conserved and variable regions, as well as long-range base-pairing interactions that bring distant parts of the RNA molecule close together (
      • Ellis J.C.
      • Brown J.W.
      The RNase P family.
      ,
      • James B.D.
      • Olsen G.J.
      • Liu J.S.
      • Pace N.R.
      The secondary structure of ribonuclease P RNA, the catalytic element of a ribonucleoprotein enzyme.
      ,
      • Gutell R.R.
      Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994.
      ,
      • Gutell R.R.
      • Larsen N.
      • Woese C.R.
      Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective.
      ,
      • Schnare M.N.
      • Damberger S.H.
      • Gray M.W.
      • Gutell R.R.
      Comprehensive comparison of structural characteristics in eukaryotic cytoplasmic large subunit (23 S-like) ribosomal RNA.
      ,
      • Seif E.R.
      • Forget L.
      • Martin N.C.
      • Lang B.F.
      Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure.
      ). Tertiary contacts, mediated by noncovalent interactions, further act as braces to tie remote parts of the secondary structure together and to form the compact three-dimensional, modular structure of the functional small subunit (SSU) and large subunit (LSU) rRNAs, as well as the RPR (
      • Wimberly B.T.
      • Brodersen D.E.
      • Clemons Jr., W.M.
      • Morgan-Warren R.J.
      • Carter A.P.
      • Vonrhein C.
      • Hartsch T.
      • Ramakrishnan V.
      Structure of the 30S ribosomal subunit.
      ,
      • Ban N.
      • Nissen P.
      • Hansen J.
      • Moore P.B.
      • Steitz T.A.
      The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution.
      ,
      • Harms J.
      • Schluenzen F.
      • Zarivach R.
      • Bashan A.
      • Gat S.
      • Agmon I.
      • Bartels H.
      • Franceschi F.
      • Yonath A.
      High resolution structure of the large ribosomal subunit from a mesophilic eubacterium.
      ,
      • Yusupov M.M.
      • Yusupova G.Z.
      • Baucom A.
      • Lieberman K.
      • Earnest T.N.
      • Cate J.H.D.
      • Noller H.F.
      Crystal structure of the ribosome at 5.5 Å resolution.
      ,
      • Schuwirth B.S.
      • Borovinskaya M.A.
      • Hau C.W.
      • Zhang W.
      • Vila-Sanjurjo A.
      • Holton J.M.
      • Cate J.H.D.
      Structures of the bacterial ribosome at 3.5 Å resolution.
      ,
      • Selmer M.
      • Dunham C.M.
      • Murphy 4th, F.V.
      • Weixlbaumer A.
      • Petry S.
      • Kelley A.C.
      • Weir J.R.
      • Ramakrishnan V.
      Structure of the 70S ribosome complexed with mRNA and tRNA.
      ,
      • Torres-Larios A.
      • Swinger K.K.
      • Krasilnikov A.S.
      • Pan T.
      • Mondragón A.
      Crystal structure of the RNA component of bacterial ribonuclease P.
      ,
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ). It is hardly conceivable that this structural and functional complexity was present at the dawn of translation and tRNA 5′-processing. So, what were the ancestral forms of the RNA components of the primordial ribosome and RNase P, and how did they evolve into their contemporary counterparts? An underlying premise that guides the evolutionary models discussed here is that a ribozyme (or a noncoding RNA) does not have to be covalently continuous to function: it may be composed of a collection of smaller “fragments” that are able to interact noncovalently to generate a three-dimensional structure that contains the functional core of its covalently continuous counterpart. We enumerate several recent findings, including the high-resolution structures of the ribosome and RNase P, that provide support for this notion and integrate insights from these observations to extend previously elaborated models of rRNA and RNase P RNA evolution (
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ,
      • Altman S.
      • Kirsebom L.A.
      Ribonuclease P.
      ).

      Ribosomal RNA

      Based on comparative structural studies of the ribosome and its constituent RNAs in many different protein-synthesizing systems, various inferences have been drawn about the origin and evolution of the ribosome and of its different functional domains (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ,
      • Mears J.A.
      • Cannone J.J.
      • Stagg S.M.
      • Gutell R.R.
      • Agrawal R.K.
      • Harvey S.C.
      Modeling a minimal ribosome based on comparative sequence analysis.
      ,
      • Smith T.F.
      • Lee J.C.
      • Gutell R.R.
      • Hartman H.
      The origin and evolution of the ribosome.
      ,
      • Bokov K.
      • Steinberg S.V.
      A hierarchical model for evolution of 23S ribosomal RNA.
      ,
      • Krupkin M.
      • Matzov D.
      • Tang H.
      • Metz M.
      • Kalaora R.
      • Belousoff M.J.
      • Zimmerman E.
      • Bashan A.
      • Yonath A.
      A vestige of a prebiotic bonding machine is functioning within the contemporary ribosome.
      ,
      • Petrov A.S.
      • Gulen B.
      • Norris A.M.
      • Kovacs N.A.
      • Bernier C.R.
      • Lanier K.A.
      • Fox G.E.
      • Harvey S.C.
      • Wartell R.M.
      • Hud N.V.
      • Williams L.D.
      History of the ribosome and the origin of translation.
      ). These studies have provided important insights into and have stimulated imaginative ideas about rRNA/ribosome evolution. Among the conclusions that are particularly germane here is the notion of modularity and domain accretion: that rRNA molecules comprise a set of discrete modules/domains that have been acquired sequentially over the evolutionary history of the ribosome (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ,
      • Smith T.F.
      • Lee J.C.
      • Gutell R.R.
      • Hartman H.
      The origin and evolution of the ribosome.
      ,
      • Bokov K.
      • Steinberg S.V.
      A hierarchical model for evolution of 23S ribosomal RNA.
      ,
      • Petrov A.S.
      • Gulen B.
      • Norris A.M.
      • Kovacs N.A.
      • Bernier C.R.
      • Lanier K.A.
      • Fox G.E.
      • Harvey S.C.
      • Wartell R.M.
      • Hud N.V.
      • Williams L.D.
      History of the ribosome and the origin of translation.
      ). Paramount among these domains, and almost certainly the oldest, is the peptidyl transferase center (PTC), located in the LSU rRNA. The accretion model (
      • Petrov A.S.
      • Gulen B.
      • Norris A.M.
      • Kovacs N.A.
      • Bernier C.R.
      • Lanier K.A.
      • Fox G.E.
      • Harvey S.C.
      • Wartell R.M.
      • Hud N.V.
      • Williams L.D.
      History of the ribosome and the origin of translation.
      ) envisages the ribosome and translation beginning with small, interacting RNAs, with the primitive rRNA growing in complexity via the incorporation of new modules. However, the evolutionary mechanism whereby contemporary SSU and LSU rRNA molecules—covalently continuous and long—might have arisen from small, noncovalently interacting RNA species has been little considered. Here, we specifically address this question.
      The discovery of ribosomes that contain fragmented rRNAs (documented in Refs.
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ) inspired the idea that contemporary long, covalently continuous rRNA molecules may have evolved from a collection of much smaller, noncovalently interacting RNA species that comprised the primordial ribosome (
      • Clark C.
      On the evolution of ribosomal RNA.
      ,
      • Boer P.H.
      • Gray M.W.
      Scrambled ribosomal RNA gene pieces in Chlamydomonas reinhardtii mitochondrial DNA.
      ). The existence of such naturally fragmented rRNAs and the demonstration that the fragmentation is inherent and not the result of degradation during sample preparation (
      • Gray M.W.
      Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.
      ,
      • Schnare M.N.
      • Gray M.W.
      Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis.
      ) indicate that covalent continuity, as seen in conventional 16S-18S SSU and 23S-28S LSU rRNAs, is not absolutely essential for function. Indeed, in protein-synthesizing cell lysates that have been treated with nuclease to remove endogenous mRNA, ribosomes contain fragmented (degraded) rRNAs but are nevertheless active in translation (
      • Kennedy T.D.
      • Hanley-Bowdoin L.K.
      • Lane B.G.
      Structural integrity of DNA and translational integrity of ribosomes in nuclease-treated cell-free protein synthesizing systems prepared from wheat germ and rabbit reticulocytes.
      ). Of course, whether a particular rRNA cleavage is deleterious depends on where these scissions occur within the rRNA structure. A single phosphodiester bond cleavage within the decoding site near the 3′-end of 16S rRNA, mediated by the bacterial cytotoxin colicin E3, is sufficient to inactivate the bacterial ribosome (
      • Senior B.W.
      • Holland I.B.
      Effect of colicin E3 upon the 30S ribosomal subunit of Escherichia coli.
      ,
      • Bowman C.M.
      • Dahlberg J.E.
      • Ikemura T.
      • Konisky J.
      • Nomura M.
      Specific inactivation of 16S ribosomal RNA induced by colicin E3 in vivo.
      ). Likewise, a single incision within the α-sarcin loop, a binding site for elongation factors (
      • Hausner T.-P.
      • Atmadja J.
      • Nierhaus K.H.
      Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors.
      ,
      • Moazed D.
      • Robertson J.M.
      • Noller H.F.
      Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA.
      ,
      • Leffers H.
      • Egebjerg J.
      • Andersen A.
      • Christensen T.
      • Garrett R.A.
      Domain VI of Escherichia coli 23 S ribosomal RNA: structure, assembly and function.
      ) that is universally conserved near the 3′-end of LSU rRNAs, is sufficient to inactivate the ribosome (
      • Endo Y.
      • Wool I.G.
      The site of action of α-sarcin on eukaryotic ribosomes: the sequence at the a-sarcin cleavage site in 28 S ribosomal ribonucleic acid.
      ,
      • García-Ortega L.
      • Álvarez-García E.
      • Gavilanes J.G.
      • Martínez-del-Pozo Á.
      • Joseph S.
      Cleavage of the sarcin–ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding.
      ). On the other hand, nonconserved regions of rRNA have been shown to be tolerant toward genetic insertions (
      • Yokoyama T.
      • Suzuki T.
      Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments.
      ), so it is hardly surprising that cleavages within naturally fragmented rRNAs are confined to these variable regions (
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ). (It is important to emphasize that although variable regions may have initially been devoid of function, the possibility remains that some may have acquired function later during ribosome evolution. For example, in some eukaryotic rRNAs, certain variable regions have become greatly enlarged into so-called expansion segments, some of which now have a regulatory function in protein synthesis (
      • Fujii K.
      • Susanto T.T.
      • Saurabh S.
      • Barna M.
      Decoding the function of expansion segments in ribosomes.
      ).)
      A model of rRNA evolution based on the idea of fragmented rRNAs has been presented and elaborated in detail elsewhere (
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ) and is briefly described here. The model envisages a stepwise evolutionary pathway (Fig. 1), with four main stages, exemplified by current examples of diverse rRNA structure. In Stage I (Fig. 1), physically separate rRNA coding modules, corresponding to discrete portions of the conserved structural core of contemporary rRNAs, are separately transcribed to generate precursor transcripts that undergo processing to generate mature 5′ and 3′ termini. The final transcripts interact noncovalently to form a primitive functional core able to catalyze peptide bond formation. Additional functional domains may have been acquired during this stage, again via noncovalent interactions. Contemporary ribosomes that exemplify features of this stage include the bipartite SSU rRNA in the mitochondrial ribosome (mitoribosome) of Euglena gracilis (
      • Spencer D.F.
      • Gray M.W.
      Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome.
      ) and, in particular, the multipartite SSU and LSU rRNAs in the mitoribosome of Plasmodium falciparum, the malaria parasite (
      • Feagin J.E.
      • Harrell M.I.
      • Lee J.C.
      • Coe K.J.
      • Sands B.H.
      • Cannone J.J.
      • Tami G.
      • Schnare M.N.
      • Gutell R.R.
      The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum.
      ), and Clathrina clathrus, a calcareous sponge (
      • Lavrov D.V.
      • Pett W.
      • Voigt O.
      • Wörheide G.
      • Forget L.
      • Lang B.F.
      • Kayal E.
      Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.
      ). In the 6-kb Plasmodium mitochondrial DNA (mtDNA), numerous short subgenic SSU and LSU rRNA coding modules are interspersed with one another and with protein-coding genes and are encoded on both strands of the mitochondrial genome. In Clathrina, reminiscent of the situation in Plasmodium, subgenic SSU and LSU rRNA coding modules are found on three of the six linear chromosomes that comprise the mitochondrial genome and are interspersed with one another and with both protein-coding and tRNA genes. In Clathrina and Plasmodium, it is clear that the rRNA subgene sets on complementary strands or separate chromosomes must be independently transcribed, with subsequent processing of primary transcripts to release the mature small rRNA pieces for mitoribosome assembly.
      Figure thumbnail gr1
      Figure 1Model for the stepwise conversion of split rRNA genes into a contiguous, single unit. RNA species 4 is depicted as containing a primitive, self-folding PTC domain to emphasize that peptide bond formation would have been supported at the earliest stages of this evolutionary pathway. MacPyMOL was used to generate the LSU rRNA structure in the center (Protein Data Bank code 1FFK; Haloarcula marismortui). Adapted from Refs.
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      and
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      . This research was originally published in Tracing Biological Evolution in Protein and Gene Structures (Gō, M., and Schimmel, P., eds). Gray, M. W., Greenwood, S. J., Smallman, D. S., Spencer, D. F., and Schnare, M. N. Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome. 1995; 65–76. © Elsevier Science B.V. and in Ribosomal RNA: Structure, Evolution, Processing, and Function in Protein Biosynthesis (Zimmermann, R. A., and Dahlberg, A. E., eds). Gray, M. W., and Schnare, M. N. Evolution of rRNA gene organization. 1996; 49–69. © CRC Press, Inc.
      In Stage II (Fig. 1), rRNA-coding modules are physically and transcriptionally linked and might be interspersed with non-rRNA-coding sequences, as in the example of the P. falciparum mtDNA. However, at this stage, all rRNA modules are co-transcribed, thus ensuring equivalent steady-state levels of the mature, noncovalently interacting products and thereby diminishing the likelihood of formation of nonfunctional, partial assemblies (a possibility in Stage I). This decisive advantage may have been the selective pressure that brought the different modules together under a single promoter. At this stage, physically linked subgenic modules are not ordered in the same 5′-to-3′ transcriptional direction as the corresponding sequences in a conventional rRNA gene; the resulting long co-transcripts, however, are processed to give small rRNA pieces, which then associate noncovalently akin to Stage I. Contemporary examples of Stage II are the bipartite mitochondrial LSU rRNA in the ciliate protozoan Tetrahymena pyriformis, where the 5′ ∼280 nt (α species) is encoded downstream of the rest of the LSU rRNA gene (β species); the α and β species are separated by a tRNALeu gene (
      • Heinonen T.Y.K.
      • Schnare M.N.
      • Young P.G.
      • Gray M.W.
      Rearranged coding segments, separated by a transfer RNA gene, specify the two parts of a discontinuous large subunit ribosomal RNA in Tetrahymena pyriformis mitochondria.
      ). An even more extreme example is highlighted in the mtDNA of the chlorophyte alga, Chlamydomonas reinhardtii, where subgenic SSU and LSU rRNA modules are scrambled and interspersed with one another and with protein-coding and tRNA genes, all co-transcribed, with post-transcriptional processing of the resulting long transcript (Fig. 2) (
      • Boer P.H.
      • Gray M.W.
      Scrambled ribosomal RNA gene pieces in Chlamydomonas reinhardtii mitochondrial DNA.
      ,
      • Boer P.H.
      • Gray M.W.
      Transfer RNA genes and the genetic code in Chlamydomonas reinhardtii mitochondria.
      ).
      Figure thumbnail gr2
      Figure 2Gene content and arrangement in the linear mitochondrial genome of the chlorophyte alga, C. reinhardtii (
      • Boer P.H.
      • Gray M.W.
      Scrambled ribosomal RNA gene pieces in Chlamydomonas reinhardtii mitochondrial DNA.
      ,
      • Boer P.H.
      • Gray M.W.
      Transfer RNA genes and the genetic code in Chlamydomonas reinhardtii mitochondria.
      ), showing the scrambled arrangement of subgenic SSU (S) and LSU (L) rRNA-coding modules, which are interspersed and co-transcribed with protein-coding and tRNA genes (W, Q, M). Protein-coding genes specify subunits of electron transport chain Complex I (nad1, nad2, nad4, nad5, and nad6), Complex III (cob), and Complex IV (cox1), as well as a reverse transcriptase-like protein (rtl). Arrows, putative promoters. SSU rRNA– and LSU rRNA–coding modules are numbered in the 5′-to-3′ transcriptional order of the corresponding sequences in their covalently continuous rRNA counterparts. Adapted from Ref.
      • Boer P.H.
      • Gray M.W.
      Transfer RNA genes and the genetic code in Chlamydomonas reinhardtii mitochondria.
      . This research was originally published in Current Genetics. Boer, P. H., and Gray, M. W. Transfer RNA genes and the genetic code in Chlamydomonas reinhardtii mitochondria. Curr. Genet. 1988; 14:583–590. © Springer.
      In Stage III (Fig. 1), rearrangements at the DNA level culminate in the standard 5′-to-3′ order of SSU and LSU rRNA sequence observed in contemporary, conventional SSU and LSU rRNAs, with SSU-coding modules physically linked to and upstream of LSU modules in the direction of transcription. The selective pressure for such rearrangement may have been the emergence of co-transcriptional folding of longer transcripts such that generation of the mature tertiary structure was facilitated and enhanced, compared with post-transcriptional assembly of separate transcripts. At this stage, rRNA transcripts retain processing sites that liberate mature rRNA segments from larger primary transcripts, so that coding modules are effectively separated by internal transcribed spacer sequences (ITSs) that are removed either co- or post-transcriptionally. The result is a ribosome in which constituent rRNAs are fragmented to an extent dependent on the number of ITSs in the primary transcript, with different parts of SSU and LSU rRNAs held together via noncovalent interactions (as in Stages I and II). Examples of Stage III are the fragmented LSU rRNAs that are found in the cytoplasmic ribosomes of kinetoplastid protozoa, such as Crithidia fasciculata (
      • Gray M.W.
      Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.
      ,
      • Spencer D.F.
      • Collings J.C.
      • Schnare M.N.
      • Gray M.W.
      Multiple spacer sequences in the nuclear large subunit ribosomal RNA gene of Crithidia fasciculata.
      ) and Trypanosoma brucei (
      • Campbell D.A.
      • Kubo K.
      • Clark C.G.
      • Boothroyd J.C.
      Precise identification of cleavage sites involved in the unusual processing of trypanosome ribosomal RNA.
      ), and especially the alga E. gracilis, whose cytoplasmic LSU rRNA exists and functions as a complex of 14 separate pieces (
      • Schnare M.N.
      • Gray M.W.
      Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis.
      ,
      • Schnare M.N.
      • Cook J.R.
      • Gray M.W.
      Fourteen internal transcribed spacers in the circular ribosomal DNA of Euglena gracilis.
      ).
      Stage IV (Fig. 1) envisages progressive loss of processing sites, which effectively converts ITSs (removed during processing) into variable regions that are now retained in the mature rRNA. Effectively, subgenic rRNA modules are “pasted together,” ultimately resulting in a collection of relatively short rRNA segments being converted over evolutionary time into a few long SSU and LSU rRNAs. We emphasize that because the Stage IV pattern of rRNA gene organization, transcription, and processing is common to all three domains of life, the evolutionary transitions we propose here must have been complete before the divergence of these domains from the LUCA. (Incidentally, this idea of converting ITSs to variable regions may be applicable in other cases of ncRNA evolution. For example, yeast U2 snRNA is 6 times larger than the mammalian counterpart due to the addition of 945 nucleotides (nt), whose deletion has no effect on growth, in sharp contrast to the two smaller domains that flank it (
      • Shuster E.O.
      • Guthrie C.
      Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides.
      ). We also refer below to similar instances with the RPR.)
      Considering that the above model of rRNA evolution was initially elaborated more than two decades ago, it is instructive to place it within the extensive body of work on ribosome structure, function, and evolution that has appeared subsequently (
      • Hsiao C.
      • Mohan S.
      • Kalahar B.K.
      • Williams L.D.
      Peeling the onion: ribosomes are ancient molecular fossils.
      ,
      • Fox G.E.
      Origin and evolution of the ribosome.
      ,
      • Wimberly B.T.
      • Brodersen D.E.
      • Clemons Jr., W.M.
      • Morgan-Warren R.J.
      • Carter A.P.
      • Vonrhein C.
      • Hartsch T.
      • Ramakrishnan V.
      Structure of the 30S ribosomal subunit.
      ,
      • Ban N.
      • Nissen P.
      • Hansen J.
      • Moore P.B.
      • Steitz T.A.
      The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution.
      ,
      • Harms J.
      • Schluenzen F.
      • Zarivach R.
      • Bashan A.
      • Gat S.
      • Agmon I.
      • Bartels H.
      • Franceschi F.
      • Yonath A.
      High resolution structure of the large ribosomal subunit from a mesophilic eubacterium.
      ,
      • Yusupov M.M.
      • Yusupova G.Z.
      • Baucom A.
      • Lieberman K.
      • Earnest T.N.
      • Cate J.H.D.
      • Noller H.F.
      Crystal structure of the ribosome at 5.5 Å resolution.
      ,
      • Schuwirth B.S.
      • Borovinskaya M.A.
      • Hau C.W.
      • Zhang W.
      • Vila-Sanjurjo A.
      • Holton J.M.
      • Cate J.H.D.
      Structures of the bacterial ribosome at 3.5 Å resolution.
      ,
      • Selmer M.
      • Dunham C.M.
      • Murphy 4th, F.V.
      • Weixlbaumer A.
      • Petry S.
      • Kelley A.C.
      • Weir J.R.
      • Ramakrishnan V.
      Structure of the 70S ribosome complexed with mRNA and tRNA.
      ,
      • Mears J.A.
      • Cannone J.J.
      • Stagg S.M.
      • Gutell R.R.
      • Agrawal R.K.
      • Harvey S.C.
      Modeling a minimal ribosome based on comparative sequence analysis.
      ,
      • Smith T.F.
      • Lee J.C.
      • Gutell R.R.
      • Hartman H.
      The origin and evolution of the ribosome.
      ,
      • Bokov K.
      • Steinberg S.V.
      A hierarchical model for evolution of 23S ribosomal RNA.
      ,
      • Krupkin M.
      • Matzov D.
      • Tang H.
      • Metz M.
      • Kalaora R.
      • Belousoff M.J.
      • Zimmerman E.
      • Bashan A.
      • Yonath A.
      A vestige of a prebiotic bonding machine is functioning within the contemporary ribosome.
      ,
      • Petrov A.S.
      • Gulen B.
      • Norris A.M.
      • Kovacs N.A.
      • Bernier C.R.
      • Lanier K.A.
      • Fox G.E.
      • Harvey S.C.
      • Wartell R.M.
      • Hud N.V.
      • Williams L.D.
      History of the ribosome and the origin of translation.
      ,
      • Frank J.
      • Agrawal R.K.
      A ratchet-like inter-subunit reorganization of the ribosome during translocation.
      ,
      • Connell S.R.
      • Takemoto C.
      • Wilson D.N.
      • Wang H.
      • Murayama K.
      • Terada T.
      • Shirouzu M.
      • Rost M.
      • Schüler M.
      • Giesebrecht J.
      • Dabrowski M.
      • Mielke T.
      • Fucini P.
      • Yokoyama S.
      • Spahn C.M.T.
      Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors.
      ). By general agreement, the most ancient part of the ribosome, and its functional heart, is the PTC, a self-folding domain in the large subunit (
      • Smith T.F.
      • Lee J.C.
      • Gutell R.R.
      • Hartman H.
      The origin and evolution of the ribosome.
      ,
      • Krupkin M.
      • Matzov D.
      • Tang H.
      • Metz M.
      • Kalaora R.
      • Belousoff M.J.
      • Zimmerman E.
      • Bashan A.
      • Yonath A.
      A vestige of a prebiotic bonding machine is functioning within the contemporary ribosome.
      ). Importantly, the PTC is effectively devoid of ribosomal proteins (
      • Hsiao C.
      • Mohan S.
      • Kalahar B.K.
      • Williams L.D.
      Peeling the onion: ribosomes are ancient molecular fossils.
      ,
      • Ban N.
      • Nissen P.
      • Hansen J.
      • Moore P.B.
      • Steitz T.A.
      The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution.
      ,
      • Selmer M.
      • Dunham C.M.
      • Murphy 4th, F.V.
      • Weixlbaumer A.
      • Petry S.
      • Kelley A.C.
      • Weir J.R.
      • Ramakrishnan V.
      Structure of the 70S ribosome complexed with mRNA and tRNA.
      ), leading to the conclusion that the ribosome is a ribozyme (
      • Cech T.R.
      The ribosome is a ribozyme.
      ,
      • Nissen P.
      • Hansen J.
      • Ban N.
      • Moore P.B.
      • Steitz T.A.
      The structural basis of ribosome activity in peptide bond synthesis.
      ). Instead of proteins, Mg2+ interactions appear to constitute the major stabilizing force within the PTC (
      • Hsiao C.
      • Mohan S.
      • Kalahar B.K.
      • Williams L.D.
      Peeling the onion: ribosomes are ancient molecular fossils.
      ,
      • Hsiao C.
      • Williams L.D.
      A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center.
      ). Another striking and universally conserved feature of the PTC is that it comprises two subregions that display 2-fold pseudosymmetry (relating to the backbone and the orientation of the subregions but not the nucleotide sequence per se) (
      • Krupkin M.
      • Matzov D.
      • Tang H.
      • Metz M.
      • Kalaora R.
      • Belousoff M.J.
      • Zimmerman E.
      • Bashan A.
      • Yonath A.
      A vestige of a prebiotic bonding machine is functioning within the contemporary ribosome.
      ,
      • Agmon I.
      • Bashan A.
      • Zarivach R.
      • Yonath A.
      Symmetry at the active site of the ribosome: structural and functional implications.
      ,
      • Zimmerman E.
      • Yonath A.
      Biological Implications of the ribosome's stunning stereochemistry.
      ,
      • Bashan A.
      • Agmon I.
      • Zarivach R.
      • Schluenzen F.
      • Harms J.
      • Berisio R.
      • Bartels H.
      • Franceschi F.
      • Auerbach T.
      • Hansen H.A.S.
      • Kossoy E.
      • Kessler M.
      • Yonath A.
      Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.
      ). These subregions provide the LSU A-site and P-site docking platforms for aminoacyl-tRNA and peptidyl-tRNA, respectively, orienting them appropriately for peptide bond formation. This primitive RNA machine may have been further stabilized initially by interactions with short RNAs complementary to the extremities of the PTC. The inference is that a primordial PTC alone could have functioned as an incipient ribosome capable of peptide bond synthesis, although it is likely that peptides or proteins must also have emerged and co-evolved with their rRNA partners very early in ribosome evolution.
      The modular construction of the ribosome is further emphasized by studies that seek to delineate a timeline for the incorporation of different functional parts into the final structure we see today. Fox (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ) has summarized cogent arguments for inferring the relative ages of different parts of the translation machinery and LSU rRNA structure as well as the temporal appearance of individual ribosomal proteins (RPs). Strategies based on RNA-RNA, RNA-protein, and protein-protein connectivity (
      • Bokov K.
      • Steinberg S.V.
      A hierarchical model for evolution of 23S ribosomal RNA.
      ,
      • Hury J.
      • Nagaswamy U.
      • Larios-Sanz M.
      • Fox G.E.
      Ribosome origins: the relative age of 23S rRNA domains.
      ) have been particularly powerful in identifying the oldest regions of the LSU rRNA, whose secondary structure is characterized by six discrete domains (I to VI). Domain V (containing the PTC) is considered to be the oldest, with Domains II and IV also being very old and likely added in that order. Notably, because Domain IV has major contacts with the SSU, the appearance of this domain likely signaled the beginning of the formation of the SSU. Remarkably, the oldest regions of the LSU rRNA identified by these approaches are largely coincident with the universally conserved LSU rRNA core identified by comparative secondary structure mapping (
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ,
      • Mears J.A.
      • Cannone J.J.
      • Stagg S.M.
      • Gutell R.R.
      • Agrawal R.K.
      • Harvey S.C.
      Modeling a minimal ribosome based on comparative sequence analysis.
      ,
      • Gutell R.R.
      Evolutionary characteristics of 16S and 23S rRNA structures.
      ).
      The evolutionary model summarized in Fig. 1 readily accommodates the notion of a timeline in the incorporation of the various structural domains in rRNA. Initially, interactions between older and newly acquired domains could have involved noncovalent bonding, principally complementary base pairing. Depending on the number of domains in question, the evolving LSU rRNA could have functioned as a collection of small, noncovalently interacting RNAs (Fig. 1). However, the model has latitude in that the conversion to a larger, covalently continuous LSU rRNA could have occurred incrementally rather than all at once. For example, localization of a Domain IV coding module upstream and in the same transcriptional orientation as a Domain V module could ultimately have led to their co-transcription, generating a Domain IV/Domain V product, with coding modules effectively separated by an ITS. This IV-ITS-V co-transcript might have been processed to excise the ITS, thereby generating the individual Domain IV and Domain V RNAs (Stage III, Fig. 1), or the ITS might have been retained, to become a variable region separating two conserved regions (Stage IV, Fig. 1). New rRNA domains could have been added in such a stepwise fashion, effectively converting initially noncovalently interacting small RNAs into a progressively larger covalently continuous species.
      One aspect of ribosome evolution to which the original model (
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ) only alluded is the necessity of co-evolution of rRNA and RPs. Although the examples cited earlier of ribosomes containing naturally fragmented rRNAs serve to underscore the idea that rRNAs do not have to be covalently continuous to function, and interacting small rRNA pieces have been demonstrated experimentally (
      • Smallman D.S.
      • Schnare M.N.
      • Gray M.W.
      RNA: RNA interactions in the large subunit ribosomal RNA of Euglena gracilis.
      ), RPs were likely important in the assembly and stabilization of ribosomes having fragmented rRNAs. As noted above, it is possible that a primordial PTC-like RNA, stabilized by interactions with Mg2+, might have been able to catalyze peptide bond synthesis in the absence of proteins; however, incorporation of additional rRNA domains likely coincided with recruitment of RPs, as well as other nonribosomal proteins, such as helicases and chaperones required in the assembly of ribosomal subunits. As with rRNA domains, a timeline in the appearance of RPs seems likely, with some ribosomal proteins arguably older than others (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ).
      Formation of the eukaryotic spliceosome is an example of how an assemblage of noncovalently interacting RNPs can be formed, emphasizing the role of various assembly proteins in the process (
      • Will C.L.
      • Lührmann R.
      Spliceosome structure and function.
      ,
      • Shi Y.
      The spliceosome: a protein-directed metalloribozyme.
      ). Pathways of ribosome assembly, including the order of the addition of RPs to rRNA, have been well worked out in a number of instances (
      • Nissen P.
      • Hansen J.
      • Ban N.
      • Moore P.B.
      • Steitz T.A.
      The structural basis of ribosome activity in peptide bond synthesis.
      ,
      • Fox G.E.
      • Naik A.K.
      The evolutionary history of the translation machinery.
      ,
      • Klein D.J.
      • Moore P.B.
      • Steitz T.A.
      The roles of ribosomal proteins in the structure assembly, and evolution of the large ribosomal subunit.
      ,
      • Shajani Z.
      • Sykes M.T.
      • Williamson J.R.
      Assembly of bacterial ribosomes.
      ,
      • Davis J.H.
      • Williamson J.R.
      Structure and dynamics of bacterial ribosome biogenesis.
      ,
      • Klinge S.
      • Woolford J.L.
      Ribosome assembly coming into focus.
      ,
      • Peña C.
      • Hurt E.
      • Panse V.G.
      Eukaryotic ribosome assembly, transport and quality control.
      ). In the specific mitochondrial cases cited earlier (P. falciparum (
      • Feagin J.E.
      • Harrell M.I.
      • Lee J.C.
      • Coe K.J.
      • Sands B.H.
      • Cannone J.J.
      • Tami G.
      • Schnare M.N.
      • Gutell R.R.
      The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum.
      ) and C. reinhardtii (
      • Boer P.H.
      • Gray M.W.
      Scrambled ribosomal RNA gene pieces in Chlamydomonas reinhardtii mitochondrial DNA.
      )), co-transcriptional assembly of the organellar ribosome is precluded by the fact that rRNA subgenic coding modules are scrambled in the mitochondrial genome and interspersed with one another and with non-rRNA genes. In these instances, long primary transcripts undergo post-transcriptional processing to yield the mature small rRNA pieces, which must then be assembled to generate the long-range noncovalent base-pairing interactions that constitute the universally conserved SSU and LSU rRNA cores. Mitochondrial RPs must participate in and help to mediate this unusual post-transcriptional assembly process. Despite the keen interest in elucidating how a functional mitoribosome containing fragmented rRNAs is cobbled together, these studies are technically challenging: pure mitochondria are often difficult to come by in large quantities, RNAs of interest are usually present in very low amounts, and the inventory of proteins (in some cases even RPs) involved in the processing/assembly pathway is incomplete. Nevertheless, the recent success in mapping the assembly path of the mitochondrial SSU in trypanosomes (
      • Saurer M.
      • Ramrath D.J.F.
      • Niemann M.
      • Calderaro S.
      • Prange C.
      • Mattei S.
      • Scaiola A.
      • Leitner A.
      • Bieri P.
      • Horn E.K.
      • Leibundgut M.
      • Boehringer D.
      • Schneider A.
      • Ban N.
      Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery.
      ) should inspire similar studies in other mitochondrial systems, given the prospects for new insights.

      RNase P

      RNase P is a Mg2+-dependent endonuclease that functions primarily in tRNA 5′ maturation in all three domains of life, with additional long noncoding RNA biogenesis-related functions in metazoans (
      • Evans D.
      • Marquez S.M.
      • Pace N.R.
      RNase P: interface of the RNA and protein worlds.
      ,
      • Ellis J.C.
      • Brown J.W.
      The RNase P family.
      ,
      • Jarrous N.
      • Gopalan V.
      Archaeal/eukaryal RNase P: subunits, functions and RNA diversification.
      ,
      • Esakova O.
      • Krasilnikov A.S.
      Of proteins and RNA: the RNase P/MRP family.
      ). The RNase P RNP employs the RPR as the catalyst to perform RNA processing (
      • Guerrier-Takada C.
      • Gardiner K.
      • Marsh T.
      • Pace N.
      • Altman S.
      The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme.
      ). It is instructive to consider some parallels between the LSU rRNA and RPR. First, akin to the rRNA, covalent continuity is not essential for RPR function (
      • Guerrier-Takada C.
      • Altman S.
      Reconstitution of enzymatic activity from fragments of M1 RNA.
      ), and even termini can be shifted, as evidenced by the near-native function of many circularly permuted RPRs (
      • Harris M.E.
      • Kazantsev A.V.
      • Chen J.L.
      • Pace N.R.
      Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.
      ). Second, long-distance, molecular struts staple independent domains in the extant RPR tertiary structures (
      • Torres-Larios A.
      • Swinger K.K.
      • Krasilnikov A.S.
      • Pan T.
      • Mondragón A.
      Crystal structure of the RNA component of bacterial ribonuclease P.
      ,
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ,
      • Lan P.
      • Tan M.
      • Zhang Y.
      • Niu S.
      • Chen J.
      • Shi S.
      • Qiu S.
      • Wang X.
      • Peng X.
      • Cai G.
      • Cheng H.
      • Wu J.
      • Li G.
      • Lei M.
      Structural insight into precursor tRNA processing by yeast ribonuclease P.
      ,
      • Wu J.
      • Niu S.
      • Tan M.
      • Huang C.
      • Li M.
      • Song Y.
      • Wang Q.
      • Chen J.
      • Shi S.
      • Lan P.
      • Lei M.
      Cryo-EM structure of the human ribonuclease P holoenzyme.
      ). Last, there is dramatic variation in RPR size with insertions of “variable” segments interspersed with conserved regions (
      • Ellis J.C.
      • Brown J.W.
      The RNase P family.
      ,
      • Kachouri R.
      • Stribinskis V.
      • Zhu Y.
      • Ramos K.S.
      • Westhof E.
      • Li Y.
      A surprisingly large RNase P RNA in Candida glabrata.
      ). Relying on these and many other recent findings from various biochemical and structural studies of RNase P, we postulate an evolutionary trajectory of RNase P that extends a model proposed two decades ago (
      • Altman S.
      • Kirsebom L.A.
      Ribonuclease P.
      ). We first provide a structural and functional context within which to appreciate how the current RPR might have emerged in four different stages (Fig. 3). In parallel with our view of rRNA evolution, the pathway we propose allows for the addition of further functional domains to RPR throughout its evolution, again initially through noncovalent interactions.
      Figure thumbnail gr3
      Figure 3Gradual conversion of the RPR from a cis-cleaving to a trans-acting ribozyme capable of processing multiple RNA substrates. MacPyMOL was used to generate the RPR depictions (Protein Data Bank code 3Q1R,
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ). Adapted in part from (
      • Altman S.
      • Kirsebom L.A.
      Ribonuclease P.
      ).
      The bacterial RPR, like protein enzymes, was shown to be modular and made up of two independently folding domains: the catalytic (C) domain that performs the canonical RNase P cleavage and the specificity (S) domain that aids in substrate recognition but is incapable of cleavage in the absence of the C domain (
      • Guerrier-Takada C.
      • Altman S.
      Reconstitution of enzymatic activity from fragments of M1 RNA.
      ,
      • Loria A.
      • Pan T.
      Domain structure of the ribozyme from eubacterial ribonuclease P.
      ,
      • Pan T.
      Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P.
      ). There are 13 universally conserved nt in RPRs from all three domains of life (
      • Evans D.
      • Marquez S.M.
      • Pace N.R.
      RNase P: interface of the RNA and protein worlds.
      ,
      • Gopalan V.
      Uniformity amid diversity in RNase P.
      ,
      • Ellis J.C.
      • Brown J.W.
      The RNase P family.
      ). These conserved nt are dispersed across five conserved regions (CR-I to CR-V) and positioned in nearly identical secondary structure locations even in RPRs of varying sizes (∼200–450 nt). Phylogenetic analyses suggest that the C domain, which contains the active site as well as CR-I, -IV, and -V, may have been the earliest embodiment of the RPR based on the available sequence data (
      • Evans D.
      • Marquez S.M.
      • Pace N.R.
      RNase P: interface of the RNA and protein worlds.
      ,
      • Siegel R.W.
      • Banta A.B.
      • Haas E.S.
      • Brown J.W.
      • Pace N.R.
      Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.
      ,
      • Sun F.J.
      • Caetano-Anollés G.
      The ancient history of the structure of ribonuclease P and the early origins of Archaea.
      ). However, the observation that a 31-nt RNA derived from Escherichia coli RPR is competent to bind and cleave pre-tRNAs (albeit inefficiently) supports the idea that the C domain may have emerged from an amalgamation of smaller modules (
      • Kikovska E.
      • Wu S.
      • Mao G.
      • Kirsebom L.A.
      Cleavage mediated by the P15 domain of bacterial RNase P RNA.
      ). Moreover, in vitro evolution experiments have yielded ∼40-nt ribozymes, indicating that RNAs of this size may suffice for ligand binding and catalysis (
      • Seelig B.
      • Jäschke A.
      A small catalytic RNA motif with Diels-Alderase activity.
      ). Such mini-RNAs have been postulated as the predecessors to macro-RNAs like the RPR (
      • Copley S.D.
      • Smith E.
      • Morowitz H.J.
      The origin of the RNA world: co-evolution of genes and metabolism.
      ). (We point out that the 31-nt RNA segment derived from E. coli RPR is absent in many archaeal and all eukaryotic RPRs.)
      In Stage I (an early RNA world setting), a primitive RPR variant was likely embedded within a larger, self-replicating genomic RNA (Fig. 3) (
      • Altman S.
      • Kirsebom L.A.
      Ribonuclease P.
      ). One model posits that an RNA akin to the tRNA acceptor–T–stem coaxial stack (including the 3′-CCA) was a tag that marked the 3′-ends and guided replication of an ancient RNA genome by ribozymes (
      • Maizels N.
      • Weiner A.M.
      Phylogeny from function: evidence from the molecular fossil record that tRNA originated in replication, not translation.
      ). If genome replication is to be uncoupled from catalysis, then all ribozymes embedded within the larger genomic RNA would have to be liberated from the larger transcript (
      • Maizels N.
      • Weiner A.M.
      Phylogeny from function: evidence from the molecular fossil record that tRNA originated in replication, not translation.
      ). By removing the genomic tag (Fig. 3A), the RPR could have played a crucial role in this regard. Such a cis-cleaving capability of the RPR is well-supported by experimental evidence. Bacterial and archaeal RPRs, either in their entirety or only the C domain, cleave covalently tethered pre-tRNAs and model stem-loop substrates (
      • Frank D.N.
      • Harris M.E.
      • Pace N.R.
      Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates.
      ,
      • Kikuchi Y.
      • Sasaki-Tozawa N.
      • Suzuki K.
      Artificial self-cleaving molecules consisting of a tRNA precursor and the catalytic RNA of RNase P.
      ,
      • Kikuchi Y.
      • Suzuki-Fujita K.
      Synthesis and self-cleavage reaction of a chimeric molecule between RNase P-RNA and its model substrate.
      ,
      • Pulukkunat D.K.
      • Gopalan V.
      Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis.
      ). In one instance, the rate of self-cleavage by an archaeal RPR C-domain–pre–tRNA conjugate was only 12-fold slower compared with the same reaction with the full-length RPR (C + S domains) (
      • Pulukkunat D.K.
      • Gopalan V.
      Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis.
      ).
      In Stage II, the need to contribute to small RNA biogenesis in trans possibly resulted in excision of the C domain from the RNA genome. If site-specific, self-cleavage of the genomic RNA resulted in smaller RNAs that needed additional 5′-processing, an efficient trans-acting RPR would have been subject to positive selection. Many studies have documented the ability of the C domain alone to catalyze 5′-processing of pre-tRNAs and model substrates in trans; the activity is weak, however, without protein cofactors (
      • Guerrier-Takada C.
      • Altman S.
      Reconstitution of enzymatic activity from fragments of M1 RNA.
      ,
      • Chen W.Y.
      • Pulukkunat D.K.
      • Cho I.M.
      • Tsai H.Y.
      • Gopalan V.
      Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex.
      ,
      • Green C.J.
      • Rivera-León R.
      • Vold B.S.
      The catalytic core of RNase P.
      ,
      • Loria A.
      • Pan T.
      The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site.
      ,
      • Tsai H.Y.
      • Pulukkunat D.K.
      • Woznick W.K.
      • Gopalan V.
      Functional reconstitution and characterization of Pyrococcus furiosus RNase P.
      ). Interestingly, these archaeal/bacterial RPR C-domain deletion derivatives foreshadowed the discovery of a shorter, naturally occurring doppelgänger: an archaeal RPR variant, lacking most of the S domain, was discovered in Thermoproteacae (
      • Lai L.B.
      • Chan P.P.
      • Cozen A.E.
      • Bernick D.L.
      • Brown J.W.
      • Gopalan V.
      • Lowe T.M.
      Discovery of a minimal form of RNase P in Pyrobaculum.
      ). This variant, as exemplified by the 208-nt version in Pyrobaculum aerophilum, supports weak tRNA 5′-maturation in vitro. Even though protein cofactors in vivo are likely obligatory for functioning of this abbreviated RPR, that it exists at all in members of the Thermoproteacae family within the crenarchaeal phylum (
      • Lai L.B.
      • Chan P.P.
      • Cozen A.E.
      • Bernick D.L.
      • Brown J.W.
      • Gopalan V.
      • Lowe T.M.
      Discovery of a minimal form of RNase P in Pyrobaculum.
      ,
      • Chan P.P.
      • Brown J.W.
      • Lowe T.M.
      Modeling the Thermoproteaceae RNase P RNA.
      ) attests to its early origins and supports the suggestion that the RPR progenitor could have been just the stand-alone C domain. The ancestral nature of the C domain is also showcased by its presence in the minimal consensus structure of the bacterial (∼210-nt) and eukaryotic (∼160-nt) RPRs that was determined by extensive phylogenetic analyses (
      • Evans D.
      • Marquez S.M.
      • Pace N.R.
      RNase P: interface of the RNA and protein worlds.
      ,
      • Siegel R.W.
      • Banta A.B.
      • Haas E.S.
      • Brown J.W.
      • Pace N.R.
      Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.
      ,
      • Marquez S.M.
      • Harris J.K.
      • Kelley S.T.
      • Brown J.W.
      • Dawson S.C.
      • Roberts E.C.
      • Pace N.R.
      Structural implications of novel diversity in eucaryal RNase P RNA.
      ). (Although there is no major difference between Stages I and II with respect to RPR function, the free-standing existence of the RPR C domain and other ribozymes would have facilitated their conversion into corresponding DNAs by reverse transcription, a key requisite for the transition from the RNA to the DNA world.)
      In Stage III (perhaps coinciding with the LUCA), adventitious interactions between the RPR C domain and another RNA likely engendered substrate-recognition payoffs. One such trans-acting RNA must have been the forerunner to the extant S domain, which has now been demonstrated in various RPRs to be critical for tRNA recognition (
      • Torres-Larios A.
      • Swinger K.K.
      • Krasilnikov A.S.
      • Pan T.
      • Mondragón A.
      Crystal structure of the RNA component of bacterial ribonuclease P.
      ,
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ,
      • Lan P.
      • Tan M.
      • Zhang Y.
      • Niu S.
      • Chen J.
      • Shi S.
      • Qiu S.
      • Wang X.
      • Peng X.
      • Cai G.
      • Cheng H.
      • Wu J.
      • Li G.
      • Lei M.
      Structural insight into precursor tRNA processing by yeast ribonuclease P.
      ,
      • Wu J.
      • Niu S.
      • Tan M.
      • Huang C.
      • Li M.
      • Song Y.
      • Wang Q.
      • Chen J.
      • Shi S.
      • Lan P.
      • Lei M.
      Cryo-EM structure of the human ribonuclease P holoenzyme.
      ,
      • Chen W.Y.
      • Pulukkunat D.K.
      • Cho I.M.
      • Tsai H.Y.
      • Gopalan V.
      Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex.
      ,
      • Loria A.
      • Pan T.
      The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site.
      ,
      • Loria A.
      • Pan T.
      Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P.
      ). However, there are no reports of these two RPR domains being transcribed from two independent genes and then assembled together (either with or without protein subunits). Even in cases where only short C domain-like RPRs have been reported (e.g. fungal mitochondria, Crenarchaea) (
      • Lai L.B.
      • Chan P.P.
      • Cozen A.E.
      • Bernick D.L.
      • Brown J.W.
      • Gopalan V.
      • Lowe T.M.
      Discovery of a minimal form of RNase P in Pyrobaculum.
      ,
      • Chan P.P.
      • Brown J.W.
      • Lowe T.M.
      Modeling the Thermoproteaceae RNase P RNA.
      ,
      • Wise C.A.
      • Martin N.C.
      Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small.
      ), there is no evidence for the presence of a free-standing RPR S domain. Combing for short, conserved stretches in S domains whose sizes range from 50 to 700 nt is difficult. We list three reasons, however, to support the likelihood of Stage III.
      First, a majority of extant RPRs have long-range, tertiary interactions that bridge the two domains and contribute to the ribozyme's stability and activity (
      • Torres-Larios A.
      • Swinger K.K.
      • Krasilnikov A.S.
      • Pan T.
      • Mondragón A.
      Crystal structure of the RNA component of bacterial ribonuclease P.
      ,
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ,
      • Lan P.
      • Tan M.
      • Zhang Y.
      • Niu S.
      • Chen J.
      • Shi S.
      • Qiu S.
      • Wang X.
      • Peng X.
      • Cai G.
      • Cheng H.
      • Wu J.
      • Li G.
      • Lei M.
      Structural insight into precursor tRNA processing by yeast ribonuclease P.
      ,
      • Wu J.
      • Niu S.
      • Tan M.
      • Huang C.
      • Li M.
      • Song Y.
      • Wang Q.
      • Chen J.
      • Shi S.
      • Lan P.
      • Lei M.
      Cryo-EM structure of the human ribonuclease P holoenzyme.
      ). These interdomain interactions, which entail tetraloop-tetraloop receptor contacts, confer weak activity on an assembly of S and C domain versions, each of which is inactive on its own (
      • Guerrier-Takada C.
      • Altman S.
      Reconstitution of enzymatic activity from fragments of M1 RNA.
      ,
      • Pan T.
      Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P.
      ).
      Second, high-resolution structures of bacterial and eukaryotic RNase P have shown that the S domains in these two RPRs (separated by billions of years in evolution) recognize the “elbow” structure that is unique to the L-shaped tRNA structure (
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ,
      • Lan P.
      • Tan M.
      • Zhang Y.
      • Niu S.
      • Chen J.
      • Shi S.
      • Qiu S.
      • Wang X.
      • Peng X.
      • Cai G.
      • Cheng H.
      • Wu J.
      • Li G.
      • Lei M.
      Structural insight into precursor tRNA processing by yeast ribonuclease P.
      ,
      • Wu J.
      • Niu S.
      • Tan M.
      • Huang C.
      • Li M.
      • Song Y.
      • Wang Q.
      • Chen J.
      • Shi S.
      • Lan P.
      • Lei M.
      Cryo-EM structure of the human ribonuclease P holoenzyme.
      ). All S domains house CR-II and CR-III, each of which forms a T-loop that consists of five consecutive nt that form a U-turn structure (
      • Chan C.W.
      • Chetnani B.
      • Mondragón A.
      Structure and function of the T-loop structural motif in noncoding RNAs.
      ). Nucleotides at positions 1 and 5 form a closing base pair, whereas position 2 stacks with position 1 and pairs with the base at position 4. The unstacked nucleobase at position 3 is used to facilitate tertiary/intermolecular contacts through either base pairing or base stacking (
      • Chan C.W.
      • Chetnani B.
      • Mondragón A.
      Structure and function of the T-loop structural motif in noncoding RNAs.
      ). The two T-loops in the RPR interdigitate and offer two unstacked bases to directly interact with the D (dihydrouridine)- and TψC-loops (
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ,
      • Lan P.
      • Tan M.
      • Zhang Y.
      • Niu S.
      • Chen J.
      • Shi S.
      • Qiu S.
      • Wang X.
      • Peng X.
      • Cai G.
      • Cheng H.
      • Wu J.
      • Li G.
      • Lei M.
      Structural insight into precursor tRNA processing by yeast ribonuclease P.
      ). The early presence of isolated S domain–like RNAs that could specifically recognize the elbow structure is supported by the salient observation that the head-to-tail interdigitated T-loop motif observed in RPRs is also used by 23S rRNA and T-box riboswitches to accomplish tRNA recognition (
      • Lehmann J.
      • Jossinet F.
      • Gautheret D.
      A universal RNA structural motif docking the elbow of tRNA in the ribosome, RNAse P and T-box leaders.
      ).
      Last, the bifurcated recognition of the tRNA substrate by RNase P has a thematic parallel in tRNA synthetases (
      • Altman S.
      • Kirsebom L.A.
      Ribonuclease P.
      ). The noncovalent interaction between the C and S domains would have been beneficial, especially if it co-evolved with the emergence of anticodon-bearing tRNAs from simple minihelices. Various arguments have been advanced to support the idea that the anticodon-D-stem stack, which allowed decoding of mRNAs, was probably added to the more ancient acceptor helix stacked on the TψC stem-loop (
      • Schimmel P.
      • Ribas de Pouplana L.
      Transfer RNA: from minihelix to genetic code.
      ,
      • Schimmel P.
      • Alexander R.
      Diverse RNA substrates for aminoacylation: clues to origins?.
      ). This adaptation was the edifice necessary to build the theater of protein synthesis. RPRs position the cleavage site by exploiting specific contacts between the C domain and the tRNA acceptor helix and 3′-CCA (when present) and between the S domain and the elbow structure (Fig. 4, A and B). Aminoacyl-tRNA synthetases have an ancient catalytic domain that recognizes the acceptor helix and an anticodon recognition domain (that was likely added later) to read the anticodon (Fig. 4C) (
      • Schimmel P.
      • Ribas de Pouplana L.
      Transfer RNA: from minihelix to genetic code.
      ,
      • Schimmel P.
      • Alexander R.
      Diverse RNA substrates for aminoacylation: clues to origins?.
      ). Thus, the transformation of mini-helices to the L-shaped tRNAs must have led to co-evolution of enzymes wherein the newer tRNA structural elements were harnessed to build specificity in recognition. Although there are no reports of independently encoded RPR S and C domains, there is an example of a split alanyl-tRNA synthetase in Nanoarchaeum equitans, where the catalytic and anticodon recognition domains are expressed from separate genes. When the two individual domains were reconstituted in vitro, the noncovalent assembly was functional (
      • Waters E.
      • Hohn M.J.
      • Ahel I.
      • Graham D.E.
      • Adams M.D.
      • Barnstead M.
      • Beeson K.Y.
      • Bibbs L.
      • Bolanos R.
      • Keller M.
      • Kretz K.
      • Lin X.
      • Mathur E.
      • Ni J.
      • Podar M.
      • et al.
      The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism.
      ).
      Figure thumbnail gr4
      Figure 4Schematic depicting thematic parallels in substrate recognition by aminoacyl-tRNA synthetases and RNase P (see “RNase P” for details). MacPymol was used to generate the RPR and tRNA depictions (Protein Data Bank Code 3Q1R,
      • Reiter N.J.
      • Osterman A.
      • Torres-Larios A.
      • Swinger K.K.
      • Pan T.
      • Mondragón A.
      Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.
      ).
      In Stage IV, rearrangements at the DNA level must have finally led to the fused arrangement (Fig. 3). Akin to the rRNA situation, various benefits likely provided the driving force for insertion of the S domain–coding sequence within that of the C domain gene. Foremost, the higher stability of the bimodular ribozyme that is assembled from a single RNA. Second, there were likely payoffs for RNA folding especially with appropriate in vivo transcriptional pause sites that allow attainment of the native fold (
      • Wong T.N.
      • Sosnick T.R.
      • Pan T.
      Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures.
      ). Third, a single gene ensures that there are no disparities in the stoichiometric amounts of the two domains synthesized. Last, the catalytic process is simplified, as the overall binding reaction is reduced from a three- to a two-component system with payoffs resulting from cooperative binding of the two domains to the substrate. A similar argument has been put forth for effective DNA recognition by two DNA-binding domains connected by a linker (
      • Klemm J.D.
      • Pabo C.O.
      Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage.
      ).

      Conclusions

      Here, we have argued that long ncRNAs such as LSU rRNA and RPR had their evolutionary origins in smaller RNAs whose genomic coding regions were subsequently “stitched together” during evolution. This concept, which thematically mirrors the genesis of multidomain proteins, likely has parallels in shaping the evolution of other ncRNAs. For example, tRNAs are generally considered to be composed of two domains, each comprising one-half of the tRNA molecule (
      • Maizels N.
      • Weiner A.M.
      Phylogeny from function: evidence from the molecular fossil record that tRNA originated in replication, not translation.
      ,
      • Noller H.F.
      On the origin of the ribosome: co-evolution of sub-domains of tRNA and rRNA.
      ,
      • Schimmel P.
      • Giegé R.
      • Moras D.
      • Yokoyama S.
      An operational RNA code for amino acids and possible relationship to genetic code.
      ,
      • Schimmel P.
      • Henderson B.
      Possible role of aminoacyl-RNA complexes in noncoded peptide synthesis and origin of coded synthesis.
      ). As described above, one domain contains the 3′-terminal CCA to which amino acids are attached; the other contains the anticodon loop that interacts with mRNA. The two domains are considered to be of different ages, with the CCA domain the older (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ). If this is the case, the two domains could well have been melded together evolutionarily by a pathway resembling the one described here for rRNA and RPR. There are other instances where this theme is not as well-defined but that merit a closer look. For example, the signal recognition particle (SRP) RNA comprises two structurally and functionally separable modules: a smaller (Alu I) domain that is believed to be involved in translational arrest and a larger domain that is essential for recognizing the nascent sequence of secretory proteins emerging from the ribosome (
      • Nagai K.
      • Oubridge C.
      • Kuglstatter A.
      • Menichelli E.
      • Isel C.
      • Jovine L.
      Structure, function and evolution of the signal recognition particle.
      ,
      • Zwieb C.
      • Bhuiyan S.
      Archaea signal recognition particle shows the way.
      ). The SRP RNA appears to have been forged from two distinct structural modules, thereby engendering functional gains, although the presence of bacterial variants that have the larger domain either without or with the smaller (Alu) domain complicates tracing the thread of evolution. (While there are examples of split transfer-mRNAs (tmRNAs) (
      • Mao C.
      • Bhardwaj K.
      • Sharkady S.M.
      • Fish R.I.
      • Driscoll T.
      • Wower J.
      • Zwieb C.
      • Sobral B.W.
      • Williams K.P.
      Variations on the tmRNA gene.
      ), we excluded them from consideration as these fragments arose from gene permutation events and differ thematically from the examples described above.)
      We have presented a portrait of ncRNAs to support the seemingly inescapable “consolidation” of domains (i.e. transition from small to large) perspective, but the counterview of “fragmentation” (large to small) also warrants consideration. For example, the evolutionary transition of ancient group II introns to the spliceosome is believed to have entailed fragmentation of the group II intron into smaller RNAs that eventually became the snRNAs capable of reassembling in trans to form the present-day splicing machinery (
      • Sharp P.A.
      “Five easy pieces”.
      ,
      • Fica S.M.
      • Tuttle N.
      • Novak T.
      • Li N.-S.
      • Lu J.
      • Koodathingal P.
      • Dai Q.
      • Staley J.P.
      • Piccirilli J.A.
      RNA catalyses nuclear pre-mRNA splicing.
      ). One can argue that fragmentation was likely advantageous in the case of the spliceosome. An early genome riddled with cis(self)-splicing introns of varying efficiency may have favored a more capable trans-splicing system, which eventually afforded other significant payoffs, including alternative splicing and the versatility afforded by fabrication of spliceosome variants with shared and distinctive components. Such flexibility would have been impossible with a single-subunit RNA, such as the group II intron from which spliceosomal snRNAs are thought to have evolved (
      • Sharp P.A.
      “Five easy pieces”.
      ,
      • Fica S.M.
      • Tuttle N.
      • Novak T.
      • Li N.-S.
      • Lu J.
      • Koodathingal P.
      • Dai Q.
      • Staley J.P.
      • Piccirilli J.A.
      RNA catalyses nuclear pre-mRNA splicing.
      ). In fact, at first glance, the existence of contemporary systems, described earlier, in which ribosomes contain naturally fragmented SSU and/or LSU rRNA species might seem to present a parallel to the spliceosome. These fragmented rRNAs undoubtedly evolved from ancestors having covalently continuous rRNAs (i.e. the fragmentation does not represent a retained ancestral trait but rather is a derived characteristic). This contention is supported by the patchy distribution of fragmented rRNAs throughout the eukaryotic or mitochondrial phylogenetic tree, in which species having fragmented rRNAs are embedded among species that have covalently continuous counterparts, which are in the majority (
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ). For both eukaryotic and mitochondrial ribosomes, we infer from the respective eukaryotic and mitochondrial phylogenetic trees that the last eukaryotic common ancestor had covalently continuous cytosolic and mitochondrial rRNAs (
      • Gray M.W.
      • Schnare M.N.
      Evolution of the modular structure of ribosomal RNA.
      ,
      • Gray M.W.
      • Greenwood S.J.
      • Smallman D.S.
      • Spencer D.F.
      • Schnare M.N.
      Ribosomal RNA in pieces: a modern paradigm of the primordial ribosome.
      ,
      • Gray M.W.
      • Schnare M.N.
      Evolution of rRNA gene organization.
      ). While the benefits (if any) of rRNA fragmentation are not immediately evident, these examples serve to emphasize the structural flexibility that is inherent in the ribosome's evolutionary history and help us to deduce the rRNA evolutionary origins. However, the selective pressures at different hierarchical levels, which balance the choice between fragmentation and consolidation, confound attempts to unambiguously map the evolutionary trajectories of the rRNA and RPR.
      While we have highlighted some unifying themes from a comparison of ribosomes and RNase P, we recognize that the molecular paleontology of these two catalytic RNPs does not permit the fashioning of a simple, linear evolutionary chronology of their catalytic RNA components. Nevertheless, the smorgasbord of ribosome and RNase P variants—wrought by chance and necessity—is arresting for its striking plurality and for highlighting the value of studying ancient and fundamental macromolecular machines in diverse organisms to better appreciate the dynamic nature of their evolution.

      Acknowledgments

      We thank Mike Ibba and Karin Musier-Forsyth (Ohio State University) for valuable input, and Hong-Duc Phan (Ohio State University) for assistance with illustrations.

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