Discovery and characterization of Acanthamoeba castellanii mitochondrial 5S rRNA

Abstract

Although 5S rRNA is a highly conserved and universal component of eubacterial, archaeal, chloroplast, and eukaryotic cytoplasmic ribosomes, a mitochondrial DNA-encoded 5S rRNA has so far been identified only in land plants and certain protists. This raises the question of whether 5S rRNA is actually required for and used in mitochondrial translation. In the protist Acanthamoeba castellanii, BLAST searches fail to reveal a 5S rRNA gene in the complete mitochondrial genome sequence, nor is a 5S-sized RNA species detectable in ethidium bromide-stained gels of highly purified mitochondrial RNA preparations. Here we show that an alternative visualization technique, UV shadowing, readily detects a novel, mitochondrion-specific small RNA in A. castellanii mitochondrial RNA preparations, and that this RNA species is, in fact, a 5S rRNA encoded by the A. castellanii mitochondrial genome. These results emphasize the need for caution when interpreting negative results that suggest the absence of 5S rRNA and/or a mitochondrial DNA-encoded 5S rRNA sequence in other (particularly protist) mitochondrial systems.

FIGURE 1.

Visualization by (A) UV shadowing and (B) ethidium bromide staining of A. castellanii cytoplasmic (cyto) and mitochondrial (mito) RNAs separated on a 10% polyacrylamide gel. Note: The low-abundance RNA in the mitochondrial RNA preparation that comigrates with cytoplasmic 5.8S rRNA has a 3′-terminal sequence identical to that of cytoplasmic 5.8S rRNA (not shown). Our data indicate that this RNA has a single 3′-terminal U residue, whereas the number of U residues is ambiguous in the published sequence (MacKay and Doolittle 1981).

FIGURE 2.

Visualization by UV shadowing of RNAs isolated from the supernatant (S100) and pellet (P100) of a 100,000g ultracentrifugation of a clarified Triton X-100 lysate of purified A. castellanii mitochondria. RNAs were separated on a 10% polyacrylamide gel. The positions of A. castellanii cytoplasmic (cyto) 5.8S and 5S rRNA markers are indicated.

FIGURE 3.

(A) Electrophoretic separation of 3′-end-labeled species X and (B) chemical sequencing of these labeled RNAs. A representative 20% polyacrylamide gel for RNA band X4 is displayed, with a portion of the deduced RNA sequence shown on the right. (C) Electrophoretic separation of 5′-end-labeled species X and (D) one-dimensional thin-layer chromatography of the products of P1 nuclease digestion of the four largest 5′-end-labeled RNAs (X1–X4). The positions of nucleoside 5′-monophosphate markers are indicated. Autoradiograms are shown in all cases.

FIGURE 4.

(A) Alignment of a conserved portion of mitochondrial (m) and eubacterial (e) 5S rRNA sequences. Sequences used (GenBank accession number in parentheses): Acanthamoeba castellanii (Aca; this work), Triticum aestivicum (Tae; M10361), Marchantia polymorpha (Mpo; M68929), Prototheca wickerhamii (Pwi; U02970), Nephroselmis olivacea (Nol; AF110138), Cyanidium caldarium (Cca; Z48930), Cyanidioschyzon merolae (Cme; D89861), Chondrus crispus (Ccr; Z47547), Pylaiella littoralis (Pli; AJ277126), Laminaria digitata (Ldi; AJ344328), Reclinomonas americana (Ram; U59762), Rhodobacter capsulata (Rca; X04585), and Escherichia coli (Eco; X00414). The spacing in the alignment represents the alternation of single- and double-stranded regions. Dashes (-) indicate alignment gaps. Numbers in parentheses indicate nucleotides not shown. The consensus (con m) indicates positions that are either identical in at least 9 of the 11 mitochondrial sequences or of the same type (Y = pyrimidine, R = purine) in all 11 mitochondrial sequences. Asterisks indicate positions in the mitochondrial consensus that are also conserved in the eubacterial sequences. (B) Potential RNA secondary structure of the longest sequence variant of the A. castellanii mitochondrial 5S rRNA. Red indicates nucleotides identical to the mitochondrial consensus. Additional potential base-pairing is indicated by broken lines. Helices I to V and loops A to E are denoted as in Burger et al. (1999).