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Alternative mRNA editing in trypanosomes is extensive and may contribute to mitochondrial protein diversity.

Ochsenreiter T, Cipriano M, Hajduk SL - PLoS ONE (2008)

Bottom Line: To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes.Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified.These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.

ABSTRACT
The editing of trypanosome mitochondrial mRNAs produces transcripts necessary for mitochondrial functions including electron transport and oxidative phosphorylation. Precursor-mRNAs are often extensively edited by specific uridine insertion or deletion that is directed by small guide RNAs (gRNAs). Recently, it has been shown that cytochrome c oxidase subunit III (COXIII) mRNAs can be alternatively edited to encode a novel mitochondrial membrane protein composed of a unique hydrophilic N-terminal sequence of unknown function and the C-terminal hydrophobic segment of COXIII. To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes. Using this data, we show that alternative editing of COXIII, ATPase subunit 6 (A6), and NADH dehydrogenase subunits 7, 8 and 9 (ND7, 8, 9) mRNAs can produce novel open reading frames (ORFs). Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified. These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms.

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Alternative editing of ND8 mRNAs.A) Protein sequence of ORF1 and ORF2, both sequences show no similarity to the bona fide ND8 (not shown). B) Bar depicts the mRNA sequence ND8-F04. Alignment of the RNA sequences for ND8, and ND8-F04 from the alternatively edited regions I, and II are shown below the bar. In black are the pre-edited residues, in grey the bona fide edited residues and in red are the alternatively edited residues from regions I–II. In black is the pre-edited, in grey the bona fide edited region and in red are the alternatively edited regions I and II. Alternative start codons UUG and GUG are shown in black and purple, respectively. Alignment of the RNA sequence of ND8 and cDNA ND8-F04 from the alternatively edited region I and II (red). Depicted above the RNA sequences are the corresponding amino acid sequences. Predicted gRNA for region I showing perfect complementarity (allowing for G:U) to ND8-F04 over 41 base pairs while having three mismatches to the ND8 sequence at positions 38 to 40 of the gRNA. Vertical bars indicate A:U or G:C base pairing; crosses indicates G:U base pairing. Red Us indicate alternatively inserted Us when compared with the bona fide ND8 sequence. Star depicts termination codon in the amino acid sequence. Underlined sequence depicts stop codon in the nucleotide sequence.
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pone-0001566-g002: Alternative editing of ND8 mRNAs.A) Protein sequence of ORF1 and ORF2, both sequences show no similarity to the bona fide ND8 (not shown). B) Bar depicts the mRNA sequence ND8-F04. Alignment of the RNA sequences for ND8, and ND8-F04 from the alternatively edited regions I, and II are shown below the bar. In black are the pre-edited residues, in grey the bona fide edited residues and in red are the alternatively edited residues from regions I–II. In black is the pre-edited, in grey the bona fide edited region and in red are the alternatively edited regions I and II. Alternative start codons UUG and GUG are shown in black and purple, respectively. Alignment of the RNA sequence of ND8 and cDNA ND8-F04 from the alternatively edited region I and II (red). Depicted above the RNA sequences are the corresponding amino acid sequences. Predicted gRNA for region I showing perfect complementarity (allowing for G:U) to ND8-F04 over 41 base pairs while having three mismatches to the ND8 sequence at positions 38 to 40 of the gRNA. Vertical bars indicate A:U or G:C base pairing; crosses indicates G:U base pairing. Red Us indicate alternatively inserted Us when compared with the bona fide ND8 sequence. Star depicts termination codon in the amino acid sequence. Underlined sequence depicts stop codon in the nucleotide sequence.

Mentions: Analysis of 78 full-length cDNAs from the ND8 gene also revealed alternatively edited mRNA. The ND8-F04 cDNA and two other identical sequences contained two regions where a total of 11 uridines are inserted alternatively at seven editing sites (Figure 2A, Table 1). This transcript contained two ORFs of similar size (Figure 2A). ORF1 started with an UUG (Leu) initiation codon 21 nucleotides downstream of the predicted 5′end and terminated with a UAA termination codon produced by alternative editing after 117 amino acids. When compared to the public databases this sequence showed no similarity to any known sequence. The second ORF (ORF2) started 47 nucleotides downstream of the 5′ end with a GUG (Val) start codon and had no termination codon in the ND8 coding sequence, however a termination codon (UAA) was found in the poly A tail which terminated the sequence after 121 amino acids. The predicted amino acid sequence (ORF2) showed weak similarity over a short stretch to a hypothetical protein from Plasmodium chabaudi (XP_744827).


Alternative mRNA editing in trypanosomes is extensive and may contribute to mitochondrial protein diversity.

Ochsenreiter T, Cipriano M, Hajduk SL - PLoS ONE (2008)

Alternative editing of ND8 mRNAs.A) Protein sequence of ORF1 and ORF2, both sequences show no similarity to the bona fide ND8 (not shown). B) Bar depicts the mRNA sequence ND8-F04. Alignment of the RNA sequences for ND8, and ND8-F04 from the alternatively edited regions I, and II are shown below the bar. In black are the pre-edited residues, in grey the bona fide edited residues and in red are the alternatively edited residues from regions I–II. In black is the pre-edited, in grey the bona fide edited region and in red are the alternatively edited regions I and II. Alternative start codons UUG and GUG are shown in black and purple, respectively. Alignment of the RNA sequence of ND8 and cDNA ND8-F04 from the alternatively edited region I and II (red). Depicted above the RNA sequences are the corresponding amino acid sequences. Predicted gRNA for region I showing perfect complementarity (allowing for G:U) to ND8-F04 over 41 base pairs while having three mismatches to the ND8 sequence at positions 38 to 40 of the gRNA. Vertical bars indicate A:U or G:C base pairing; crosses indicates G:U base pairing. Red Us indicate alternatively inserted Us when compared with the bona fide ND8 sequence. Star depicts termination codon in the amino acid sequence. Underlined sequence depicts stop codon in the nucleotide sequence.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215773&req=5

pone-0001566-g002: Alternative editing of ND8 mRNAs.A) Protein sequence of ORF1 and ORF2, both sequences show no similarity to the bona fide ND8 (not shown). B) Bar depicts the mRNA sequence ND8-F04. Alignment of the RNA sequences for ND8, and ND8-F04 from the alternatively edited regions I, and II are shown below the bar. In black are the pre-edited residues, in grey the bona fide edited residues and in red are the alternatively edited residues from regions I–II. In black is the pre-edited, in grey the bona fide edited region and in red are the alternatively edited regions I and II. Alternative start codons UUG and GUG are shown in black and purple, respectively. Alignment of the RNA sequence of ND8 and cDNA ND8-F04 from the alternatively edited region I and II (red). Depicted above the RNA sequences are the corresponding amino acid sequences. Predicted gRNA for region I showing perfect complementarity (allowing for G:U) to ND8-F04 over 41 base pairs while having three mismatches to the ND8 sequence at positions 38 to 40 of the gRNA. Vertical bars indicate A:U or G:C base pairing; crosses indicates G:U base pairing. Red Us indicate alternatively inserted Us when compared with the bona fide ND8 sequence. Star depicts termination codon in the amino acid sequence. Underlined sequence depicts stop codon in the nucleotide sequence.
Mentions: Analysis of 78 full-length cDNAs from the ND8 gene also revealed alternatively edited mRNA. The ND8-F04 cDNA and two other identical sequences contained two regions where a total of 11 uridines are inserted alternatively at seven editing sites (Figure 2A, Table 1). This transcript contained two ORFs of similar size (Figure 2A). ORF1 started with an UUG (Leu) initiation codon 21 nucleotides downstream of the predicted 5′end and terminated with a UAA termination codon produced by alternative editing after 117 amino acids. When compared to the public databases this sequence showed no similarity to any known sequence. The second ORF (ORF2) started 47 nucleotides downstream of the 5′ end with a GUG (Val) start codon and had no termination codon in the ND8 coding sequence, however a termination codon (UAA) was found in the poly A tail which terminated the sequence after 121 amino acids. The predicted amino acid sequence (ORF2) showed weak similarity over a short stretch to a hypothetical protein from Plasmodium chabaudi (XP_744827).

Bottom Line: To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes.Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified.These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.

ABSTRACT
The editing of trypanosome mitochondrial mRNAs produces transcripts necessary for mitochondrial functions including electron transport and oxidative phosphorylation. Precursor-mRNAs are often extensively edited by specific uridine insertion or deletion that is directed by small guide RNAs (gRNAs). Recently, it has been shown that cytochrome c oxidase subunit III (COXIII) mRNAs can be alternatively edited to encode a novel mitochondrial membrane protein composed of a unique hydrophilic N-terminal sequence of unknown function and the C-terminal hydrophobic segment of COXIII. To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes. Using this data, we show that alternative editing of COXIII, ATPase subunit 6 (A6), and NADH dehydrogenase subunits 7, 8 and 9 (ND7, 8, 9) mRNAs can produce novel open reading frames (ORFs). Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified. These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms.

Show MeSH
Related in: MedlinePlus