Complete set of mitochondrial pan-edited mRNAs in Leishmania mexicana amazonensis LV78

Abstract

Editing of mRNA transcribed from the mitochondrial cryptogenes ND8 (G1), ND9 (G2), G3, G4, ND3 (G5), RPS12 (G6) was investigated in Leishmania mexicana amazonensis, strain LV78, by amplification of the cDNA, cloning and sequencing. For each of these genes, extensively and partially edited transcripts were found to be relatively abundant compared to the respective pre-edited molecules. Moreover, the editing patterns observed in a majority of transcripts of each gene were consistent among themselves which allowed for inferring consensus editing sequences. The open reading frames contained in the consensus sequences were predicted to encode polypeptides that were highly similar to their counterparts in other species of Trypanosomatidae. Several kinetoplast DNA minicircles from this species available in the public domain were found to contain genes for guide RNAs which mediate editing of some of the mRNAs. The results indicate that the investigated strain of L. m. amazonensis has preserved its full editing capacity in spite of the long-term maintenance in culture. This property differs drastically from the other Leishmania species which lost some or all of the G1-G5 mRNA editing ability in culture.

Fig. 1

Comparison of MURF1 and MURF5 amino acid sequences from Leishmania mexicana amazonensis (Lam) with their homologs from Leishmania donovani (Ld), Leishmania tarentolae (Lt), Phytomonas serpens (Ps), and Trypanosoma brucei (Tb). Multiple sequence alignment was generated using Vector NTI. Identical residues found in a given position in all sequences are highlighted with yellow, and those in the majority of sequences are indicated with blue, while conservative substitutions are shown with green. Dashes represent alignment gaps: (A) multiple alignment of the complete predicted MURF5 sequences; (B); alignment of the N-terminal ends of the MURF1 sequences.

Fig. 2

Results of RT-PCR amplification of mitochondrial cryptogene transcripts from promastigotes of L. m. amazonensis after electrophoresis in a 2% (w/v) agarose gel. Each cryptogene, indicated above the panel, was analyzed in two reactions: lanes indicated with R represent RT-PCR with mitochondrial RNA as the source of material; lanes labeled with D represent standard amplification reactions using kDNA. Dots indicate the bands representing pre-edited cDNA amplification products (with sizes matching the respective genomic fragments), arrows show positions of presumptive fully edited cDNA products. Positions of 1 kb DNA Ladder bands (Invitrogen) are shown to the right.

Fig. 3

Pan-edited mitochondrial ND8 (NADH dehydrogenase subunit 8) mRNA sequence of L. m. amazonensis. The sequence (Lam, upper lane) and its in silico translation (the lane underneath the mRNA sequence) are compared with the respective protein and mRNA sequences from L. tarentolae LEM125 (Lt). Edited regions on both mRNA are underlined. Unedited 5′ and 3′ sequences expected to be present in Lam mRNA and used as PCR primers are shown with bold italics. Encoded nucleotides preserved in the edited transcripts are shown with upper case letters; inserted uridylates are shown with low case u’s and deleted uridylates are shown with asterisks. Dashes represent alignment gaps. The exact 5′ and 3′ ends of the Lam mRNA are unknown, while the actual ends of the Lt mRNA are as shown.

Fig. 4

Pan-edited mitochondrial ND9 (NADH dehydrogenase subunit 9) mRNA sequence of L. m. amazonensis. Most designations are as in Fig. 3. The 3′-end alternative editing pattern caused by the presence of an extra U (highlighted with black) in some Lam transcripts (see the text) is also shown. A minicircle-encoded guide RNA, gND9-V, is shown matched with the Lam edited sequence with canonical base pairs indicated with vertical lines and non-canonical G-U base pairs indicated with colons.

Fig. 5

Pan-edited mitochondrial G3 mRNA sequence of L. m. amazonensis. The Lam sequence is compared with the G3 edited sequence of Phytomonas serpens (Ps). A minicircle-encoded gRNA, mediating the last editing block, is also shown. Other designations are as in Fig. 3 and 4.

Fig. 6

Pan-edited mitochondrial G4 mRNA and protein sequences of L. m. amazonensis: (A) comparison of the edited G4 mRNA and the derived amino acid sequence with the T. brucei sequences (Tb). Two minicircle-encoded gRNAs, gG4-I and gG4-IV, are also shown; (B) multiple alignment of the G4 sequences from the two aforementioned species and L. tarentolae (Lt). The remaining designations are as in Fig. 1, 3 and 4.

Fig. 6

Pan-edited mitochondrial G4 mRNA and protein sequences of L. m. amazonensis: (A) comparison of the edited G4 mRNA and the derived amino acid sequence with the T. brucei sequences (Tb). Two minicircle-encoded gRNAs, gG4-I and gG4-IV, are also shown; (B) multiple alignment of the G4 sequences from the two aforementioned species and L. tarentolae (Lt). The remaining designations are as in Fig. 1, 3 and 4.

Fig. 7

Pan-edited mitochondrial ND3 (NADH dehydrogenase subunit 3) mRNA and protein sequences of L. m. amazonensis: (A) comparison with the edited ND3 mRNA and the derived amino acid sequences from L. tarentolae LEM125 (Lt). The unmatched U-residue in the Lt sequence which is responsible for the relative reading frame shift is highlighted with black. A minicircle-encoded gRNA, gND3-IV, is also shown; (B) multiple alignment of the ND3 amino acid sequences. Other designations are as in Fig. 1, 3 and 4.

Fig. 7

Pan-edited mitochondrial ND3 (NADH dehydrogenase subunit 3) mRNA and protein sequences of L. m. amazonensis: (A) comparison with the edited ND3 mRNA and the derived amino acid sequences from L. tarentolae LEM125 (Lt). The unmatched U-residue in the Lt sequence which is responsible for the relative reading frame shift is highlighted with black. A minicircle-encoded gRNA, gND3-IV, is also shown; (B) multiple alignment of the ND3 amino acid sequences. Other designations are as in Fig. 1, 3 and 4.

Fig. 8

Pan-edited mitochondrial RPS12 (ribosomal protein S12) mRNA sequence of L. m. amazonensis. The Domain Connections Sequences DCS-I and DCS-II are boxed. Other designations are as in Fig. 3.