Mitochondrial RNAs of myxomycetes terminate with non-encoded 3' poly(U) tails

Abstract

We examined the 3' ends of edited RNAs from the myxomycetes Stemonitis flavogenita and Physarum polycephalum using a modified anchor PCR approach. Surprisingly, we found that poly(A) tails are missing from the cytochrome c oxidase subunit 1 mRNA (coI) from both species and the cytochrome c oxidase subunit 3 mRNA (cox3) from P. polycephalum. Instead, non-encoded poly(U) tails of varying length were discovered at the 3' ends of these transcripts. These are the first described examples of 3' poly(U) tails on mature mRNAs in any system.

Figure 1

Poly(U) tails on S.flavogenita coI cDNA. The 3′ untranslated region of S.flavogenita coI cDNA clones are aligned with directly sequenced DNA PCR product (GenBank accession no. AF239222). The alignment begins with nucleotide 1805 (T) in the GenBank record, and the inferred stop codon is underlined. Dotted regions within clone sequences indicate identity with the DNA sequence. The regions with white letters inside a black box indicate poly(U) tails. The gray shaded regions designate the primer used for reverse transcription. Slight variation in the length of poly(G) regions of reverse transcription primers may be due to minor imperfections in the primer pool, or PCR slippage through the homopolymeric region. The final base of the primer is a non-C anchor base, intended to direct annealing location of this primer to the most 5′ end of a potentially long poly(G) run during reverse transcription. Non-primer-derived nucleotides beyond the poly(U) tails (black letters on a white background within the clone sequences) are probably artifacts of the tailing proceedure, as explained in the Results.

Figure 2

Poly(U) tails on P.polycephalum coI cDNA. The 3′ untranslated region of P.polycephalum coI cDNA clones are aligned with DNA sequence from Jonatha Gott (personal communication). Numerical notation is continuous with GenBank accession no. L14779. The inferred stop codon is underlined. Annotation as in Figure 1.

Figure 3

Poly(U) tails on P.polycephalum cox3 cDNA. The 3′ untranslated region of P.polycephalum cox3 cDNA clones are aligned with corresponding DNA sequence (GenBank accession no. AF084526). The first nucleotide of the alignment corresponds to nucleotide 3368 (T) in the GenBank record, and the inferred stop codon is underlined. Annotation as in Figure 1.

Figure 4

Physarum polycephalum mitochondrial large subunit ribosomal RNA (mit LSU) cDNA and nuclear small subunit ribosomal RNA (SSU) cDNA. (A) Physarum polycephalum mitochondrial large subunit ribosomal RNA (mit LSU) cDNA clones are aligned with the DNA sequence (GenBank accession no. AF080602). The first position of the alignment corresponds to nucleotide 2783 (T) in the GenBank record. (B) Physarum polycephalum nuclear small subunit ribosomal RNA (SSU) cDNA clones are aligned with the DNA sequence (GenBank accession no. X13160). The alignment begins with nucleotide 1950 (G) in the GenBank record. Annotation as in Figure 1. (The extreme ends of the lower clones’ reverse transcription primer sequences have been omitted for clarity.)