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Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences.

Ivanov IP, Firth AE, Michel AM, Atkins JF, Baranov PV - Nucleic Acids Res. (2011)

Bottom Line: We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences.Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes.In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

View Article: PubMed Central - PubMed

Affiliation: BioSciences Institute, University College Cork, Cork, Ireland. iivanov@genetics.utah.edu

ABSTRACT
In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized--both for increased coding capacity and potentially also for novel regulatory mechanisms--remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

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Plots showing density of mRNA fragments protected by ribosomes for NM_004494 and NM_001010858. The position of the annotated AUG codon was taken as zero; relative coordinates of stop codons and predicted non-AUG initiators are indicated. Regions corresponding to annotated CDSs are highlighted in dark grey; regions corresponding to non-AUG-initiated extensions are highlighted in light grey. The presence of ribosomal footprints in the region of an extension indicates that the initiation of translation takes place upstream of the annotated CDS.
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Figure 7: Plots showing density of mRNA fragments protected by ribosomes for NM_004494 and NM_001010858. The position of the annotated AUG codon was taken as zero; relative coordinates of stop codons and predicted non-AUG initiators are indicated. Regions corresponding to annotated CDSs are highlighted in dark grey; regions corresponding to non-AUG-initiated extensions are highlighted in light grey. The presence of ribosomal footprints in the region of an extension indicates that the initiation of translation takes place upstream of the annotated CDS.

Mentions: The second set of experimental data supporting our predictions was obtained from recently published ribosomal profiling experiments carried out in human cells (43). In this work, mRNA fragments protected by ribosomes were converted into a library of oligonucleotides and subjected to massively parallel sequencing. The alignment of corresponding sequences onto sequences of mRNAs allows detection of ribosomal locations and the density of the ribosomes on mRNA. We have aligned raw sequences obtained by Guo et al. (43) using the bowtie program (44) and quantified locations of the ribosomes on the mRNA sequences where non-AUG initiation was predicted (see ‘Materials and Methods’ section). We have been able to extract reads for 32 of 59 genes. Examples of ribosomal profiles for NM_004494 and NM_001010858 are shown in Figure 7. For the other sequences, the number of footprints corresponding to CDS and non-AUG extensions, as well as ribosomal density in these regions, are given in Table 1. The presence of ribosomal profiles in the extensions upstream of annotated CDSs indicates that initiation takes place upstream of annotated AUG initiation codons and supports our predictions regarding non-AUG initiation.Figure 7.


Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences.

Ivanov IP, Firth AE, Michel AM, Atkins JF, Baranov PV - Nucleic Acids Res. (2011)

Plots showing density of mRNA fragments protected by ribosomes for NM_004494 and NM_001010858. The position of the annotated AUG codon was taken as zero; relative coordinates of stop codons and predicted non-AUG initiators are indicated. Regions corresponding to annotated CDSs are highlighted in dark grey; regions corresponding to non-AUG-initiated extensions are highlighted in light grey. The presence of ribosomal footprints in the region of an extension indicates that the initiation of translation takes place upstream of the annotated CDS.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105428&req=5

Figure 7: Plots showing density of mRNA fragments protected by ribosomes for NM_004494 and NM_001010858. The position of the annotated AUG codon was taken as zero; relative coordinates of stop codons and predicted non-AUG initiators are indicated. Regions corresponding to annotated CDSs are highlighted in dark grey; regions corresponding to non-AUG-initiated extensions are highlighted in light grey. The presence of ribosomal footprints in the region of an extension indicates that the initiation of translation takes place upstream of the annotated CDS.
Mentions: The second set of experimental data supporting our predictions was obtained from recently published ribosomal profiling experiments carried out in human cells (43). In this work, mRNA fragments protected by ribosomes were converted into a library of oligonucleotides and subjected to massively parallel sequencing. The alignment of corresponding sequences onto sequences of mRNAs allows detection of ribosomal locations and the density of the ribosomes on mRNA. We have aligned raw sequences obtained by Guo et al. (43) using the bowtie program (44) and quantified locations of the ribosomes on the mRNA sequences where non-AUG initiation was predicted (see ‘Materials and Methods’ section). We have been able to extract reads for 32 of 59 genes. Examples of ribosomal profiles for NM_004494 and NM_001010858 are shown in Figure 7. For the other sequences, the number of footprints corresponding to CDS and non-AUG extensions, as well as ribosomal density in these regions, are given in Table 1. The presence of ribosomal profiles in the extensions upstream of annotated CDSs indicates that initiation takes place upstream of annotated AUG initiation codons and supports our predictions regarding non-AUG initiation.Figure 7.

Bottom Line: We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences.Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes.In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

View Article: PubMed Central - PubMed

Affiliation: BioSciences Institute, University College Cork, Cork, Ireland. iivanov@genetics.utah.edu

ABSTRACT
In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized--both for increased coding capacity and potentially also for novel regulatory mechanisms--remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

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