A surprisingly large RNase P RNA in Candida glabrata

Abstract

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

FIGURE 1.

The putative RNA expressed in C. glabrata. (A) Northern blot. An oligonucleotide complementary to the conserved P4 helix region was used to probe the total RNA of S. cerevisiae (Sc) and C. glabrata (Cg). The position of tRNAs and 18S and 25S rRNA is shown by nonspecific binding or ethidium bromide staining. (B) RT-PCR. The total RNA of C. glabrata or S. cerevisiae was reverse-transcribed with CAGL3 primer, and the DNA was amplified by PCR with CAGL5 and CAGL3 as primers (see Materials and Methods). The DNA markers (M) were shown as 2.0, 1.2, and 0.7 kb, and so on. The 1.1-kb product was sequenced to be the right DNA coding for the putative whole-length P RNA. The small band (~300 bp) was determined to be a nonspecific product from an mRNA (CAGL0L06116g, putative protein similar to YGL185c of S. cerevisiae).

FIGURE 2.

The putative RNA is in the RNase P enzyme complex. The nuclear RNase P enzyme from C. glabrata was purified by an ion exchange column. (Lane 2) The initial sample; (lanes 3–15) fractions eluted from the column; (lanes 1,16) controls or markers. (A) The enzyme activity assay of the fractions. The enzyme complex was eluted by ~350 mM KCl from anion exchange column, similar to that of S. cerevisiae enzyme and human enzyme. (Lane 1) Control of the pre-tRNA alone; (lane 16) the pre-tRNA processed by Escherichia coli enzyme. The products of #11 and #16 had the exact same length when separated by a denaturing 12% high-resolution gel (data not shown). (B) RT-PCR reactions of the fractions. (Lane 1) The DNA markers are shown as 2.0, 1.0, and 0.5 kb, and so on; (lane 16) total RNA of C. glabrata.

FIGURE 3.

Secondary structure consensus of the conserved core of hemiascomycete RNase P RNAs. Domain I is the Specificity domain and domain II the Catalytic domain. The colored lines represent helices. The color code corresponds to that in the alignment (Fig. 4). Only five helices (P2, P3a, P4, P7, P10/11) have a constant length in the hemiascomycetes. Gray thick lines represent single strands. Black thin lines indicate junctions between two segments containing no nucleotide and necessary for the drawing. The black thin line arrows indicate the 5′ → 3′ direction. Letters beside the lines indicate conserved nucleotides among the hemiascomycete species. CR I (Conserved Region) to CR V are five well-conserved sequences among the three domains of life (Chen and Pace 1997). Because the consensus sequences are longer, we note them CR I-HY to CR V-HY (HY for Hemiascomycete Yeasts). The consensus nomenclature follows that of Cornish-Bowden (1985) with an uppercase letter when it concerns 100% of sequences and a lowercase when there is a difference in less than three sequences among the 37 sequences of the alignment. The nucleotide abbreviations are (N) any nucleotide; (R) A or G; (Y) U or C; (B) C, G, or U; (D) A, G, or U; (M) A or C; (W) A or U. Large arrows indicate sequence insertions in at least one hemiascomycete species. Red large arrows indicate sequence insertions in C. glabrata and sometimes in other species. Helix nomenclature comes from the homologous helix nomenclature of bacteria (Haas et al. 1994). Helices with unsettled homology were named eP, for “eukaryal Paired” region (Frank et al. 2000). (See Table 2 for variation types found in P RNAs of each species.)

FIGURE 4.

Hemiascomycete sequence alignment of the conserved segments of the RNase P RNA structure (helices with conserved lengths and the conserved regions CR I-HY to CR V-HY as shown in Fig. 3). The complete alignment can be obtained from the authors. The names and the conserved sequences are shown on the first two lines. On the next two lines are displayed the names of helices with the bracket notation (a dot indicates a single-stranded nucleotide and brackets, open for the 5′-end and closed for the 3′-end, indicate helices). For the other lines, sequences of each species are aligned in a phylogenetic order (Kurtzman and Robnett 1998, 2003; Dujon et al. 2004). The first column indicates species names (for abbreviations, see Table 1). The first sequence of C. guilliermondii (Cgui1) was found in the RNase P database with a synonymous name Pichia guilliermondii. The second sequence (Cgui2) was found by PCR amplification and has a few sequence variations, especially in CR III-HY. The archiascomycete S. pombe sequence is indicated here as an external reference, and the consensus does not include it. In each sequence, a dash sign (—) indicates a 1-nt gap and a gap of several nucleotides is noted by (. . .). Red underlined pairs of nucleotides indicate that they do not form either a Watson-Crick or a GoU wobble pair. Bulges are highlighted in light gray and terminal loops in dark gray. Some nucleotides are highlighted in light blue to emphasize the variations that occur in only few hemiascomycetes. P RNA sequences of other species were found in the RNase P database (Brown 1999), but were omitted here because identity percentage with the aligned sequences are higher than 95%. Sequences of Saccharomyces diastaticus has 99.64% identity (id.) with S. cerevisiae, Saccharomyces bayanus has 100% id. with Saccharomyces uvarum, S. bayanus has 99.27% id. with S. cerevisiae, and Saccharomyces carlsbergensis has 97.49% id. with Saccharomyces pastorianus (this last difference occurs only in the P1 helix). Furthermore, S. diastaticus is another name for the S. cerevisiae species; S. carlsbergensis for the S. pastorianus species; and S. uvarum and S. bayanus for the S. globosus species (Kurtzman and Fell 1998). However, paradoxically, the S. globosus sequence is quite different from the S. bayanus sequence (100% id. with S. uvarum), which is almost identical to the S. cerevisiae sequence. Moreover, the S. globosus sequence presents a P10a insertion, while neither S. bayanus nor S. cerevisiae presents such an insertion. The C. stellatoidea sequence has 98.71% identity with that of C. krusei.