SEA-PHAGES | Follow-up Clarifying Question about tRNA and protein genes not overlapping

Link to this post \| posted 02 May, 2023 03:58
fbaliraine	In my previous post entitled “Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?” (https://seaphages.org/forums/topic/5365/) I thought this question was settled. I want to delete the gene in phage Glaske16 at position 60940-61320 bp, but because several recent annotations have kept it, and it has more than 70 hits to the HNH endonuclease in phagesDB, I am seeking a second opinion on this. Its sequence is MQREYMRRWVANRRSAFFASKQCAMCGAGEELELDHIDPTKKVDHRIWSWTDARRSEELAKCQVLCASCHKKKTGEQWYANRSVSENAHHGTSRRYRKMKCRCGLCRLGNTNRSRALRQRHRVPVE Despite more than 70 hits to HNH endonuclease in phagesDB, this gene has low (<50%) coding potential in Genemark_S, and entirely no CP in Genemark_smeg, and TB. THIS GENE OVERLAPS 15bp WITH A tRNA CALLED BY ARAGORN v1.2.41 AND tRNA-SE v. 2.0. WITH AN INFERNAL SCORE OF 55.5. I am asking this question because of the statement from the Resource Guide entitled, “Predicting tRNA and tmRNA genes” (https://seaphagesbioinformatics.helpdocsonline.com/article-40): “It is highly unusual that a phage tRNA willi) Be encoded within an ORF called by Glimmer and GeneMark that has high coding potential, (ii) Be encoded on the opposite strand as a number of other phage tRNAs found in the same genome, (iii) Be encoded at a genomically distant location from the other tRNA genes in a genome. In general, violation of any of the three preceding conditions is sufficient for exclusion of a potential tRNA from an annotation (we have found a single high scoring tRNA that is not part of the rest of the large cluster, however this situation is very rare).” Whereas I am inclined to delete the protein coding gene and keep the tRNA since it is called by ARAGORN and tRNAscan-SE with a high infernal score (55.5), I also note that this tRNA is distant from other tRNAs, being 387 bp apart, which seems to violate caveat iii above. According to the forum post, “How close can one pack protein and tRNA's genes” of Feb 24, 2016, Dr Pope stated that, “We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close.” I realize that there may be exceptions though but wanted to be sure. Some M1 phages such as Reindeer & Iphrane7 do not have this gene but have the “Glu” tRNA (61306-61380 bp in Glaske16; see figures & DNA Master file attached), and we know that tRNAs tend to be conserved. Edited 02 May, 2023 04:16 346Kb 67Kb

Link to this post | posted 02 May, 2023 03:58

In my previous post entitled “Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?” (https://seaphages.org/forums/topic/5365/) I thought this question was settled. I want to delete the gene in phage Glaske16 at position 60940-61320 bp, but because several recent annotations have kept it, and it has more than 70 hits to the HNH endonuclease in phagesDB, I am seeking a second opinion on this. Its sequence is MQREYMRRWVANRRSAFFASKQCAMCGAGEELELDHIDPTKKVDHRIWSWTDARRSEELAKCQVLCASCHKKKTGEQWYANRSVSENAHHGTSRRYRKMKCRCGLCRLGNTNRSRALRQRHRVPVE

Despite more than 70 hits to HNH endonuclease in phagesDB, this gene has low (<50%) coding potential in Genemark_S, and entirely no CP in Genemark_smeg, and TB. THIS GENE OVERLAPS 15bp WITH A tRNA CALLED BY ARAGORN v1.2.41 AND tRNA-SE v. 2.0. WITH AN INFERNAL SCORE OF 55.5.
I am asking this question because of the statement from the Resource Guide entitled, “Predicting tRNA and tmRNA genes” (https://seaphagesbioinformatics.helpdocsonline.com/article-40):
“It is highly unusual that a phage tRNA will smile

i) Be encoded within an ORF called by Glimmer and GeneMark that has high coding potential, (ii) Be encoded on the opposite strand as a number of other phage tRNAs found in the same genome, (iii) Be encoded at a genomically distant location from the other tRNA genes in a genome. In general, violation of any of the three preceding conditions is sufficient for exclusion of a potential tRNA from an annotation (we have found a single high scoring tRNA that is not part of the rest of the large cluster, however this situation is very rare).”

Whereas I am inclined to delete the protein coding gene and keep the tRNA since it is called by ARAGORN and tRNAscan-SE with a high infernal score (55.5), I also note that this tRNA is distant from other tRNAs, being 387 bp apart, which seems to violate caveat iii above.
According to the forum post, “How close can one pack protein and tRNA's genes” of Feb 24, 2016, Dr Pope stated that, “We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close.”

I realize that there may be exceptions though but wanted to be sure. Some M1 phages such as Reindeer & Iphrane7 do not have this gene but have the “Glu” tRNA (61306-61380 bp in Glaske16; see figures & DNA Master file attached), and we know that tRNAs tend to be conserved.

Edited 02 May, 2023 04:16

Link to this post \| posted 02 May, 2023 12:36
debbie	Hi Fred, I think that you have covered all of the bases in this evaluation. I would avoid calling this gene. The instances where I know there is a tRNA in a predicted protein do not look like this one. However, when I saw the hit to an HNH that was confounding. However, I don't think it is an HNH. The H-N-H seems missing to me. I would call call the protein, but include the tRNAs. I would also draw attention to this in your cover sheet. The QCer will review all of this to confirm. Best, debbie

Link to this post | posted 02 May, 2023 12:36

debbie

Hi Fred,
I think that you have covered all of the bases in this evaluation. I would avoid calling this gene. The instances where I know there is a tRNA in a predicted protein do not look like this one. However, when I saw the hit to an HNH that was confounding. However, I don't think it is an HNH. The H-N-H seems missing to me. I would call call the protein, but include the tRNAs. I would also draw attention to this in your cover sheet. The QCer will review all of this to confirm.
Best,
debbie

Link to this post \| posted 04 May, 2023 23:11
fbaliraine	Thank you, Debbie! I will keep the gene with its tRNA overlap and push it over to QCer as you have suggested. I have tried searching for an article that would directly document evidence of overlaps between tRNA and protein-coding genes but was unsuccessful, although the Wright et al (2022) article (https://doi.org/10.1038/s41576-021-00417-w) has some documentation of overlaps between non-coding RNA (ncRNA) and protein-coding genes. Fred

Link to this post | posted 04 May, 2023 23:11

fbaliraine

Thank you, Debbie!
I will keep the gene with its tRNA overlap and push it over to QCer as you have suggested. I have tried searching for an article that would directly document evidence of overlaps between tRNA and protein-coding genes but was unsuccessful, although the Wright et al (2022) article (https://doi.org/10.1038/s41576-021-00417-w) has some documentation of overlaps between non-coding RNA (ncRNA) and protein-coding genes.
Fred

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Follow-up Clarifying Question about tRNA and protein genes not overlapping