*3.3.2. Stop codon translation after frameshift: ND6*

For gene ND6, the stopless ORF annotated in GenBank does not align with any ND6-like protein. This conundrum is solved by Blastp analysis of the peptide translated from the +1 frameshifted sequence of ND6 as it is annotated in GenBank. It aligns with 86% similarity with the mitochondrial NADH dehydrogenase subunit 6 of congeneric *Aleurodicus dugesii* (positions 33–137 in *Aleurodicus dispersus* and 32–129 in *Aleurodicus dugesii*, e value 1 × 10−<sup>37</sup>, not shown). Hence, the annotated gene corresponds to a stopless frame that does not translate into a recognizable mitochondrial protein, while the +1 frame, which contains three stop codons codes for ND6. Only one of the stop codons is within the alignment, where it corresponds to a tyrosine in *Aleurodicus dugesii*. This implies translation of at least one stop codon, as previously described for other short mitochondrial protein coding genes where the protein coding region includes a programmed frameshift and translation of stops (ND3 in birds [76] and in turtles [77]). It seems plausible that ND6 translation starts in the 5′ region of the frame as annotated in GenBank, and then a programmed frameshift occurs in the vicinity of the 5′-starting point of the alignment. Translation of the stop codon by tyrosine is compatible with translation by tRNAs with near-cognate anticodons [78–80].

*3.3.4. Stop codon depletion in antisense strand: ND1*

tRNAs [82]).

The annotation in GenBank for ND1 does not correspond to a protein homologous to NADH dehydrogenase subunit 1. However, the peptide translated from the +1 frame of the opposite (antisense) strand has high homology with NADH dehydrogenase subunit 1 from *Aleurodicus dugesii* (YP\_026064, 94% similarity for the complete length, e value 0). This implies regular encoding of that protein. The misannotation originates from depletion of all stops in one frame of the antisense strand of that gene. The corresponding antisense frame has 15 TAR stop codons in the regular ND1 of *Aleurodicus dugesii*. **Figure 3** aligns the peptide translated from this presumably noncoding antisense frame in *Aleurodicus dugesii* with the peptide translated from the GenBank-annotated frame. Stop codons correspond in this alignment mainly to serine (seven cases), then to tryptophan (two cases) and once each to leucine, lysine, methionine, and asparagine. This predicted translation is to much lower extents compatible with near cognate translation, and might be due to specific tRNA(s) with anticodon(s) matching stops. The fact that this antisense frame is stop codon depleted in *Aleurodicus dispersus* so that it does not necessitate any special translational machinery for its expression suggests the possibility that this frame is translated in *Aleurodicus dugesii* (and in other species) and produces an unknown functional protein, and this is due to stop codon translation by antiterminator tRNAs. Indeed, the entirety of both mitochondrial strands is transcribed to RNA; hence, RNA corresponding to this supposedly noncoding strand necessarily exists and could be translated [81]. The alignment suggests that the amino acid most probably inserted by that stop-suppressor tRNA is serine. This is in line with previous observations from cytochrome c oxidase subunit I from the silkworm *Samia ricini* [7, 9], where stop codons in a usually noncoding frame systematically mutated to serine. This finding strengthens serine as the likely residue inserted at stop codons in insect mitochondria. This awaits confirmation by translation and tRNA aminoacylation experiments (as for example done for giant virus

Directed Mutations Recode Mitochondrial Genes: From Regular to Stopless Genetic Codes

http://dx.doi.org/10.5772/intechopen.80871

63

**Figure 3.** Alignment between the peptide translated from the +1 frameshifted antisense sequence of ND1 in *Aleurodicus dugesii* (YP\_026063) and the peptide translated from the ORF annotated in GenBank for ND1 in *Aleurodicus dispersus* (JX566506). Underlined asterisks indicate stops in the antisense sequence of *Aleurodicus dugesii*, which frequently

correspond to serine in the stop-depleted ORF of *Aleurodicus dispersus.*

#### *3.3.3. Frameshift with stop translation: CytB*

The situation in CytB is similar and reminds again known cases of proteins coded by two frames. The ORF as annotated in GenBank has high homology (96% similarity) from residue 137 to 355 with the regular cytochrome B of *Aleurodicus dugesii* (YP\_026063, e value 5 × 10−91, **Figure 2**). The 5′ extremity of cytochrome B is coded by the +1 frameshifted sequence of the gene, as indicated by high similarity (88%) in the alignment from residues 6–136 with the regular cytochrome B of *Aleurodicus dugesii* (YP\_026063, e value 5 × 10−<sup>54</sup>, **Figure 2**). Position 131 is a stop that corresponds to a tyrosine in *Aleurodicus dugesii*. Hence, this gene's coding structure implies frameshift and probable stop translation, potentially by near cognate anticodon.

**Figure 1.** Alignment between peptide translated from the +1 frameshifted annotated GenBank sequence of gene AT8 in JX566506 with the protein translated from sequence AGA54141. Bold indicates similar residues, and underlined letters are for identical residues.


**Figure 2.** Alignment between peptides translated from frame 0 and the +1 frameshifted annotated GenBank sequence of gene CytB in JX566506 with *Aleurodicus dugesii* cytochrome B, sequence YP\_026063. Bold indicates similar residues, and underlined letters are for identical residues. Frame +1 of sequence annotated in GenBank JX566506.

#### *3.3.4. Stop codon depletion in antisense strand: ND1*

frameshifted sequence of ND6 as it is annotated in GenBank. It aligns with 86% similarity with the mitochondrial NADH dehydrogenase subunit 6 of congeneric *Aleurodicus dugesii* (positions 33–137 in *Aleurodicus dispersus* and 32–129 in *Aleurodicus dugesii*, e value 1 × 10−<sup>37</sup>, not shown). Hence, the annotated gene corresponds to a stopless frame that does not translate into a recognizable mitochondrial protein, while the +1 frame, which contains three stop codons codes for ND6. Only one of the stop codons is within the alignment, where it corresponds to a tyrosine in *Aleurodicus dugesii*. This implies translation of at least one stop codon, as previously described for other short mitochondrial protein coding genes where the protein coding region includes a programmed frameshift and translation of stops (ND3 in birds [76] and in turtles [77]). It seems plausible that ND6 translation starts in the 5′ region of the frame as annotated in GenBank, and then a programmed frameshift occurs in the vicinity of the 5′-starting point of the alignment. Translation of the stop codon by tyrosine is compatible

The situation in CytB is similar and reminds again known cases of proteins coded by two frames. The ORF as annotated in GenBank has high homology (96% similarity) from residue 137 to 355 with the regular cytochrome B of *Aleurodicus dugesii* (YP\_026063, e value 5 × 10−91, **Figure 2**). The 5′ extremity of cytochrome B is coded by the +1 frameshifted sequence of the gene, as indicated by high similarity (88%) in the alignment from residues 6–136 with the regular cytochrome B of *Aleurodicus dugesii* (YP\_026063, e value 5 × 10−<sup>54</sup>, **Figure 2**). Position 131 is a stop that corresponds to a tyrosine in *Aleurodicus dugesii*. Hence, this gene's coding structure implies frameshift and probable stop translation, potentially by near cognate anticodon.

**Figure 1.** Alignment between peptide translated from the +1 frameshifted annotated GenBank sequence of gene AT8 in JX566506 with the protein translated from sequence AGA54141. Bold indicates similar residues, and underlined letters

**Figure 2.** Alignment between peptides translated from frame 0 and the +1 frameshifted annotated GenBank sequence of gene CytB in JX566506 with *Aleurodicus dugesii* cytochrome B, sequence YP\_026063. Bold indicates similar residues, and

underlined letters are for identical residues. Frame +1 of sequence annotated in GenBank JX566506.

with translation by tRNAs with near-cognate anticodons [78–80].

*3.3.3. Frameshift with stop translation: CytB*

62 Mitochondrial DNA - New Insights

are for identical residues.

The annotation in GenBank for ND1 does not correspond to a protein homologous to NADH dehydrogenase subunit 1. However, the peptide translated from the +1 frame of the opposite (antisense) strand has high homology with NADH dehydrogenase subunit 1 from *Aleurodicus dugesii* (YP\_026064, 94% similarity for the complete length, e value 0). This implies regular encoding of that protein. The misannotation originates from depletion of all stops in one frame of the antisense strand of that gene. The corresponding antisense frame has 15 TAR stop codons in the regular ND1 of *Aleurodicus dugesii*. **Figure 3** aligns the peptide translated from this presumably noncoding antisense frame in *Aleurodicus dugesii* with the peptide translated from the GenBank-annotated frame. Stop codons correspond in this alignment mainly to serine (seven cases), then to tryptophan (two cases) and once each to leucine, lysine, methionine, and asparagine. This predicted translation is to much lower extents compatible with near cognate translation, and might be due to specific tRNA(s) with anticodon(s) matching stops.

The fact that this antisense frame is stop codon depleted in *Aleurodicus dispersus* so that it does not necessitate any special translational machinery for its expression suggests the possibility that this frame is translated in *Aleurodicus dugesii* (and in other species) and produces an unknown functional protein, and this is due to stop codon translation by antiterminator tRNAs. Indeed, the entirety of both mitochondrial strands is transcribed to RNA; hence, RNA corresponding to this supposedly noncoding strand necessarily exists and could be translated [81]. The alignment suggests that the amino acid most probably inserted by that stop-suppressor tRNA is serine. This is in line with previous observations from cytochrome c oxidase subunit I from the silkworm *Samia ricini* [7, 9], where stop codons in a usually noncoding frame systematically mutated to serine. This finding strengthens serine as the likely residue inserted at stop codons in insect mitochondria. This awaits confirmation by translation and tRNA aminoacylation experiments (as for example done for giant virus tRNAs [82]).


**Figure 3.** Alignment between the peptide translated from the +1 frameshifted antisense sequence of ND1 in *Aleurodicus dugesii* (YP\_026063) and the peptide translated from the ORF annotated in GenBank for ND1 in *Aleurodicus dispersus* (JX566506). Underlined asterisks indicate stops in the antisense sequence of *Aleurodicus dugesii*, which frequently correspond to serine in the stop-depleted ORF of *Aleurodicus dispersus.*
