Alternative Proteoform Prediction

Multiple translation initiation sites (TISs) may allow for alternative proteoforms to arise from the translation of just a single mRNA. RNA-Seq is limited in that it cannot be utilized to predict alternative proteoforms, and the standard proteomics approaches alone may prove insufficient. In this regard, Ribosome profiling (Ribo-Seq) could be advantageous when used in conjunction with other techniques, by profiling initiating ribosomes that are enriched at TISs. Here, we’ve selected some research articles that discuss alternative proteoform prediction and feature ribosome profiling, highlighting the unique insights gained.

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

Nucleic Acids Research, 2011; 39(10), pp.4220–4234
Ivanov, I.P., Firth, A.E., Michel, A.M., Atkins, J.F. and Baranov, P.V.

Generally, translation initiation has been understood to occur at the AUG codon closest to the messenger RNA 5’ cap in eukaryotes. However, it is now known that initiation can arise from codons differing from AUG by a single nucleotide i.e. CUG, GUG, AUA. Such non-AUG initiation has been confirmed in several human genes, however, further investigation is required to scope the breadth of this occurrence in humans. In this research article the authors develop and utilize a phylogenetic-based method to predict in-frame translation within 5’ untranslated regions of orthologous genes, and in turn, expand upon previously published coding sequence annotations. This was achieved through the use of evolutionary signatures of protein-coding sequences to gauge translation initiation upstream of annotated sequences.

Key Findings

    • Novel conserved potential non-AUG-initiated N-terminal extensions were observed in 42 human genes.
    • Known non-AUG-initiated extensions were also found to be conserved in 17 other genes.
    • Non-AUG-initiated products described in existing publications and ribosome profiling datasets were also verified via independent experimentation.


Further investigation is needed to examine the extent of non-AUG initiation in mammals, as alternative initiation regulation has the potential to coordinate alternative proteoforms at a global level. This research also highlights the advantages of using comparative genomics to predict conserved N-terminal extensions that may otherwise go unnoticed. However, while it has been shown that a translational regulation mechanism can exploit variation of start codon selection, the extent of its utilization in humans remains to be seen. Expanding upon the known conserved non-AUG initiation sites in humans would prove beneficial.

N-terminal Proteomics and Ribosome Profiling Provide a Comprehensive View of the Alternative Translation Initiation Landscape in Mice and Men

Molecular & Cellular Proteomics, 2014; 13(5), pp.1245-1261
Van Damme, P., Gawron, D., Van Criekinge, W. and Menschaert, G.

Recorded non-annotated or alternative translation initiation sites (aTIS) have been greatly underestimated, which has been made known to the wider community through recently published ribosome profiling analyses. In higher eukaryotes, approximately one fifth of all known protein N-termini are resulting from initiation at near-cognate start codons, or from alternative splicing. Proteome diversity has the potential for expansion as alternative proteoforms possessing different N-terminal extensions or truncations may exhibit other functions. This can be brought about by in-frame translation within the 5’ leader or leaky scanning resulting in translation from downstream in-frame AUG codons, as well as non-AUG initiation codons.

Key Findings

  • At least one-fifth of known protein N termini point to aTIS, and the authors detail upwards of 1,700 distinct alternative proteoforms in humans and mice, in addition to 4500 database annotated protein N termini.
  • Previously published ribosome profiling datasets were used to generate customized databases to illustrate the use of initiator methionine decoded near-cognate start codons.
  • Several novel aTIS were verified experimentally via mutagenesis, and meta-analyses showed that these reside in strong Kozak-like motifs. These aTIS are conserved in eukaryotes, which may suggest a biological relevance.
  • TargetP analysis was used to predict that aTIS frequently result in predicted altered subcellular localization patterns.


Approximately one-third of the novel protein N-termini described in this article were associated with alternative translation initiation. The authors suggest that the use of ribosome profiling strategies alongside proteomic approaches will aid in providing a more comprehensive mapping of TIS, which have the potential to exhibit alternative functional translation products.

GWIPS-viz as a tool for exploring ribosome profiling evidence supporting the synthesis of alternative proteoforms

Proteomics, 2015; 15(14), pp.2410-2416
Michel, A.M., Ahern, A.M., Donohue, C.A. and Baranov, P.V.

Utilizing sequence information alone may have significant limitations when reporting translation initiation sites (TISs), even with the use of phylogenetic data. Standard proteomics strategies have certain shortfalls as the N-termini of synthesized proteins are frequently processed. Ribosome profiling (Ribo-Seq) may prove a more beneficial approach in terms of locating and examining TISs, as this method provides a snapshot of ribosome occupancy at a transcriptome-wide level. The GWIPS‐viz (Genome Wide Information on Protein Synthesis visualized) browser is a freely available tool that facilitates access to genomic alignments of published Ribo-Seq and mRNA-Seq data including the related annotation tracks. In this article, the authors describe the potential uses for GWIPS-viz, such as examining ribosome occupancy at 5’ ends of protein coding genes, which allows for the investigation of AUG and non-AUG TISs associated with alternative proteoforms.

Key Findings

  • Ribo‐Seq tracks for several organisms can be displayed, which permits cross‐species comparison of orthologous genes.
  • Ribosome profiling datasets from a multitude of laboratories are available on the browser, which can be used to evaluate the reproducibility of the ribosome densities.
  • GWIPS‐viz can also be used to assess the TISs associated with proteoforms truncated at the N‐terminus, especially when initiation at the downstream TIS is significantly more efficient i.e. the human PRKAA1 (Protein Kinase AMP-Activated Catalytic Subunit Alpha 1) gene.
  • Additionally, this browser can be used to identify the translated ORFs that differ to the one corresponding to the CDS.


GWIPS‐viz has proven to be a useful tool for making predictions, particularly for those who require Ribo-Seq-generated experimental evidence of alternative proteoforms to confirm analyses made with other approaches.

Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

eLife, 2020, 147(4); eLife 2020;9:e52611
Wangen, J.R. and Green, R.

Stop codon readthrough (SCR) is the phenomenon in which a ribosome reaches a stop codon (either UAG, UAA or UGA) and instead of terminating translation and releasing the completed protein it miscodes the stop codon and proceeds to continue decoding until it arrives at another stop codon. This can be caused by interference from certain chemicals which inhibit the ribosome’s ability to detect stop codons, one such example would be the aminoglycoside G418. Here in this paper, the authors used ribosome profiling to study the effect of treating mammalian cells with G418 and how it impacted the ribosome’s ability to recognize  stop codons.

Key Findings

  • Using a dual-luciferase reporter assay, the authors investigated the ability of various aminoglycosides (AG) to promote readthrough of a premature termination codon (PTC). Of the six AG’s used only G418 and gentamicin were capable of potently stimulating PTC readthrough.
  • Using ribosome profiling, the authors found that the peak of terminating ribosomes decreased in cells treated with G418 and that there was a much higher ribosome density found in the 3’ leader of cells treated with G418 than other AGs.
  • In addition, the authors examined the effects of AG treatment on translation initiation and elongation. G418 appeared to be the most disruptive for both phases of translation and caused an increase in the ribosome occupancy at initiation codons.
  • One mRNA of particular interest encodes the enzyme AMD1, which is critical for determining the balance between polyamines and S-adenosylmethionine levels in the cell. When cells were treated with G418, AMD1 exhibited the largest changes in mRNA levels and RPF (ribosome protected fragment) density.


This papers shows how the activities of aminoglycosides promote readthrough of stop codons at a genome wide level. There appears to be an evolutionary pressure for genes to select more efficient stop codons that are more capable of resisting readthrough. As a number of genetic conditions result from incorrect stop codons in genes (like cystic fibrosis), these findings will help in finding drugs that may be potentially useful as treatment for these genetic conditions.

Systematic analysis of the PTEN 5′ leader identifies a major AUU initiated proteoform

Open Biology, 2016; 6(5), pp.150203
Tzani, I., Ivanov, I.P., Andreev, D.E., Dmitriev, R.I., Dean, K.A., Baranov, P.V., Atkins, J.F. and Loughran, G.

Since the first development of ribosome profiling, reports of translation within the 5′ leaders in humans continue to surface. There is a general consensus that the encoded peptide is of less significance, however, recently published datasets have permitted the prediction of 5’ leader translation. This was brought about by means of phylogenetic detection of sequences within 5’ leaders that have appeared under coding restraint. Based on this, these authors previously reported a CUG-initiated N-terminal extension to PTEN, a tumor suppressor in humans. In this research article, they expanded upon their investigation of this PTEN 5’ leader and the associated translational events.

Key Findings

  • At least two additional non-AUG-initiated PTEN proteoforms were identified and these were found to be expressed in the majority of the human cell lines tested.
  • The most abundant extended PTEN proteoform initiates at a conserved AUU codon and extends the canonical AUG-initiated PTEN by 146 amino acids.
  • All of the N-terminally extended PTEN proteoforms that were tested retain the ability to downregulate the PI3K pathway.
  • It was verified experimentally that translation of two conserved AUG-initiated upstream open reading frames within the PTEN 5′ leader regulate the ratio of PTEN proteoforms.


The analyses described in this article, together with the investigations carried out on PTEN from other researchers, could have a significant impact on the interpretation of previously published material on PTEN, in terms of catalytic activity, localization, and polymorphisms within the 5’ leader. There is also a potential for production of chemotherapeutic agents based on the identification of the novel PTEN proteoforms described here.

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