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. 2005 May;11(5):739-51.
doi: 10.1261/rna.7211705. Epub 2005 Apr 5.

Structural implications of novel diversity in eucaryal RNase P RNA

Steven M Marquez  1 , J Kirk Harris, Scott T Kelley, James W Brown, Scott C Dawson, Elisabeth C Roberts, Norman R Pace
Affiliations

Structural implications of novel diversity in eucaryal RNase P RNA

Steven M Marquez et al. RNA. 2005 May.

Abstract

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.

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Figures

FIGURE 1.
FIGURE 1.
RNA molecules isolated from individual gradient DEAE-Toyopearl fractions. (A) RNAs isolated from DEAE-Toyopearl fractions were 3′-end labeled with [5′-32P]pCp by T4 RNA ligase, and separated on a 6% denaturing polyacrylamide gel. M (RNA markers); (Eco) 3′-end labeled E. coli RNase P RNA. Black arrows identify A. castellanii RNAs that roughly coincide with enzyme activity. Approximate molecular sizes are indicated. White arrows identify RNAs that did not correlate with activity and were used as negative controls in primer extension experiments. (B) A. castellanii RNase P activity distribution from the column shown in panel A. Samples from the column shown in panel A were assayed with 32P-pre-tRNA as described in Materials and Methods. The products were resolved on a 6% sequencing gel. The upper band in the autoradiogram is the substrate (pre-tRNA) and the lower band is the product (mature-tRNA). (C) Primer extension analysis of putative G. intestinalis RNase P RNA. Primer extension by avian myeloblastosis virus reverse transcriptase was carried out with 32P-labeled oligonucleotide G120R and purified G. intestinalis RNAs as described in Materials and Methods, and the products were resolved on a 6% sequencing gel. RNAs that served as templates are as follows: lanes, (+) in vitro transcribed putative G. intestinalis RNase P RNA; primer extension products using RNAs purified from DEAE-Toyopearl fractions 1–21.
FIGURE 2.
FIGURE 2.
Secondary structure of A. castellanii RNase P RNA. Helices are labeled 5′ to 3′, P1–P19 as defined for bacterial RNase P RNA (Haas et al. 1994). Nucleotides circled in black are universally conserved among all three domains of life and constitute the five Conserved Regions I–V. Base pairs indicated by closed dots indicate a conserved noncanonical (G•U or A•C) interaction. The A. castellanii RNase P RNA secondary structure is based on evidence provided by an alignment of 63 diverse eucaryal RNase P RNA sequences. Shaded boxes indicate base pairs supported by a Hij score ≥32 (χ2 test, P < 0.0002; 9 d.f.). Outlined base pairs indicate base pairs confirmed by covariation among eucaryal RNase P RNAs. Bold nucleotides indicate the location of the weak consensus sequence identified in J3a3b of eucaryal RNase P RNAs. Lines connecting circled bases indicate tertiary interactions supported by statistical covariation analysis. The structure of the P12 helix is not established by comparative results and so is represented by a dashed circle. The 5′ and 3′ ends of the A. castellanii RNase P RNA, as well as all new sequences in this study, have not been experimentally determined. Instead, the terminus of helix P1 is arbitrarily established as the end of complementarity.
FIGURE 3.
FIGURE 3.
Compensatory length variation. The X-axis represents eucaryal RNase P RNA sequences as numbered in the eucaryal RNase P 63 alignment (http://pacelab.colorado.edu/publications.html/). The Y-axis is the length, in nucleotides, of the J5/7/8 and J5/7/CR IV regions and helix P12 as indicated.
FIGURE 4.
FIGURE 4.
J3a/3b consensus sequence. Nucleotide conservation is measured in bits of information. The information contained in a nucleotide position ranges from zero to 2 bits. One hundred percent conserved positions contain two bits of information while completely random positions contain zero bits of information. Horizontal-axis numbers indicate nucleotide position in J3a/3b (A. castellanii numbering). Sequence logo generated by using “WebLogo” at http://www.weblogo.berkeley.edu/.
FIGURE 5.
FIGURE 5.
Phylogenetic distribution of eucaryal RNase P RNA sequences. Representative organism groups from the major eucaryotic kingdoms are shown as a diagrammatic phylogenetic tree. The phylogenetic tree is based on small subunit ribosomal RNA sequences and is modified from Dawson and Pace (2002). Bold names indicate phylogenetic kingdoms represented by RNase P RNA sequences. Boxed names represent taxa from which eucaryal RNase P RNA sequences were determined in this study.
FIGURE 6.
FIGURE 6.
Secondary structures of native eucaryal RNase P RNAs and with nonhomologous sequences omitted. The secondary structures of (A) S. cerevisiae and (B) G. intestinalis with and without J5/7/8, J8/9, J9/10,11, P12, and P19. Arrows and numbers indicate positions and size, respectively, deleted to form minimal structures.
FIGURE 7.
FIGURE 7.
Phylogenetic minimum consensus RNase P RNA secondary structures. Updated phylogenetic bacterial minimum consensus RNase P RNA secondary structure (Siegel et al. 1996) and the eucaryal minimum consensus RNase P RNA secondary structure are shown.
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