FUS gene is dual-coding with both proteins united in FUS-mediated toxicity

Marie A. Brunet, Jean-Francois Jacques, Sonya Nassari, Giulia E. Tyzack, Philip McGoldrick, Lorne Zinman, Steve Jean, Janice Robertson, Rickie Patani, Xavier Roucou

Posted on: 12 May 2020 , updated on: 13 May 2020

Preprint posted on 14 April 2020

One gene, two proteins: Is FUS a case of Dr. Jekyll and Mr. Hyde?

Selected by Madhuja Samaddar

Categories: cell biology, molecular biology, neuroscience

Background:

The term proteome defines the set of proteins known to be expressed from a fixed set of genes, typically the genome of an organism. The concept of ‘one gene one polypeptide’ has been long revised to include our understanding of processes such as alternative splicing and ribosomal frameshifting, the latter best known as a strategy employed by viruses to increase proteomic complexity from a limited set of genes [1]. Additional modes of non-conventional gene expression in both prokaryotes and eukaryotes, with the potential to vastly increase proteomic complexity are now increasingly appreciated. Thus conventional genome annotations relying on criteria like a single open reading frame (ORF) per transcript and a minimum codon length, are now challenged by the concept of alternative ORFs. These alternative ORFs can give rise to functional protein products by a variety of mechanisms including cap-independent translation [2], translation from RNAs previously designated as non-coding [3], or additional translation upstream or downstream of the annotated ORFs. The use of alternative start codons is a strategy common to many examples of alternative ORFs [4].

In this exciting new preprint, Brunet et al. report the identification of an alternative ORF in the FUS coding sequence which encodes a protein they call altFUS, and show that it may at least be partially to blame for FUS-associated toxicity. FUS is a nuclear-resident RNA-binding protein whose variants demonstrate altered cytoplasmic localization and aggregation, characteristically linked to diseases such as amyotrophic lateral sclerosis (ALS), and rare forms of frontotemporal dementia (FTD) [5].

Key Findings:

Utilizing the OpenProt resource they had previously developed [6], the authors explored the coding potential of the FUS gene to identify a predicted frame shifted ORF, resulting in a 169 amino acid protein. The specific transcript for this predicted coding protein, altFUS, was found to be highly abundant in brain tissue. Mining of existing ribosome profiling data revealed accumulation of initiating ribosomes at the altFUS initiating methionine, indicating actual translation of this alternative ORF. Several unique altFUS peptides were identified from proteomic datasets, allowing the generation of a custom antibody to detect altFUS. This enabled the specific detection of altFUS expression, which was remarkably observed not just in cell lines but pathological tissue samples and iPSC-derived motor neurons. Further, altFUS was found to be a mitochondria-localized protein (Figure 1), capable of altering the mitochondrial network, and acting cooperatively with FUS in reducing mitochondrial membrane potential. Based on insights from protein-protein interaction pathways, the authors investigated the contribution of altFUS to cellular stress and autophagy. Interestingly, they found that altFUS promotes assembly of the characteristic cytoplasmic aggregates seen in disease-linked FUS mutants; and that it alone can inhibit autophagy in the absence of FUS.

Figure 1. The mitochondrial localization of altFUS (Reproduced from the preprint by under a CC-BY 4.0 international license).

Mutations in FUS that were previously believed to be synonymous and potentially harmless, were found to instead affect the altFUS coding sequence, leading to enhanced TDP-43 aggregation, another pathological hallmark of ALS. Finally, using an in vivo Drosophila model of neurodegeneration, the authors show that while altFUS alone could not recapitulate the disease phenotype, both FUS and altFUS expression are required for the toxicity reflected by loss of motor neuron activity.

Why I picked this preprint:

This work by Brunet et al. is a good example of how our progress with understanding disease mechanisms could be limited by the existing knowledge of players involved. In this study, altFUS is found to be not just endogenously expressed but also at least partially responsible for the alterations in physiology, previously attributed to FUS. Further, mutations previously believed to be synonymous in the context of FUS, take on new meaning in case of altFUS. The contribution of alternative gene expression to disease pathology is already an expanding field, particularly in our understanding of various neurodegenerative conditions. The best known example of this is Huntington’s Disease where toxic polypeptides of variable lengths arise from the expansion of trinucleotide repeats (encoding poly-glutamine stretches) on the HTT (Huntingtin) gene. Mounting evidence for additional non-conventional modes of translation opens up the possibility of a complement of unannotated proteins with potentially unknown physiological functions: a new ‘dark proteome’ [7].

References for further reading:

Programmed Ribosomal Frameshifting Goes Beyond Viruses. (2006) doi: 10.1128/microbe.1.521.1
Cap-Independent Translation: What’s in a Name? (2018) https://doi.org/10.1016/j.tibs.2018.04.011
Translation and functional roles of circular RNAs in human cancer. (2020) https://doi.org/10.1186/s12943-020-1135-7
Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape. (2015) https://doi.org/10.1371/journal.pgen.1005641
TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. (2010) https://doi.org/10.1016/S1474-4422(10)70195-2
OpenProt: a more comprehensive guide to explore eukaryotic coding potential and proteomes. (2019) https://doi.org/10.1093/nar/gky936
Alternative ORFs and small ORFs: shedding light on the dark proteome. (2019) https://doi.org/10.1093/nar/gkz734

Questions for the authors:

What fraction of known disease-linked proteins do you think possess alternative ORFs? Do specific sequence features e.g. intrinsically disordered regions represent a higher likelihood of leading to alternative ORFs?
altFUS is expressed even under normal conditions and in control tissues, but could specific conditions alter the stoichiometry of expression of the two proteins? If both are necessary for cooperative toxicity, then could altered stoichiometry be a natural mechanism to counteract toxicity under normal conditions?
Does altFUS possess a canonical mitochondrial targeting sequence or does it associate with mitochondria only under certain conditions (e.g. α-synuclein associates with mitochondria under conditions of stress)? This could help to understand whether altFUS has normal physiological functions at the mitochondria regardless of its pathological contributions.

Tags: alternative orfs, amyotrophic lateral sclerosis, dark proteome, fus, gene expression

doi: https://doi.org/10.1242/prelights.20492

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Author's response

Xavier Roucou and Marie A. Brunet shared

1. What fraction of known disease-linked proteins do you think possess alternative ORFs? Do specific sequence features e.g. intrinsically disordered regions represent a higher likelihood of leading to alternative ORFs?

Answer – This is pretty much the one-million-dollars question here! First, the number of known proteins associated to diseases is constantly growing, but even if we were to know the definite number, it would be difficult to predict which proportion of the genes encoding such disease-related proteins have an alternative ORF (altORF). In a previous work [1], we have seen that human mRNAs hold on average 3.6 ORFs; whether all of these are translated is unlikely but remains to be explored.

Intrinsically disordered regions are indeed associated with dual-coding events [2,3]. Our lab continues to explore whether other motifs or features could indicate the presence of a dual-coding region.

2. altFUS is expressed even under normal conditions and in control tissues, but could specific conditions alter the stoichiometry of expression of the two proteins? If both are necessary for cooperative toxicity, then could altered stoichiometry be a natural mechanism to counteract toxicity?

Answer – This is a very interesting question. The stoichiometry is pivotal to the correct assembly and function of proteins, so it is very likely that an altered stoichiometry between FUS and altFUS might either amplify or counteract toxicity. Previous studies have shown that the translational landscape changes with environmental stresses, thus privileging the translation of one ORF on a transcript with multiple ORFs [4,5]. Additionally, novel sources of regulation of translation might also emerge as the field of altORFs grows. As both in-depth and large-scale studies of altORFs are published, a functional cooperation is often observed between proteins encoded on the same mRNA [1,6]. It is likely that the stoichiometry of these proteins is regulated and that changes to the stoichiometry would alter their function.

3. Does altFUS possess a canonical mitochondrial targeting sequence or does it associate with mitochondria only under certain conditions (e.g. α-synuclein associates with mitochondria under conditions of stress)? This could help to understand whether altFUS has normal physiological functions at the mitochondria regardless of its pathological contributions.

Answer – Interestingly, altFUS does not have a canonical mitochondrial targeting sequence, but it is predicted to be at the mitochondria using bioinformatics tools (mostly due to its enrichment in charged residues). As we can see in Figure 2, altFUS is mainly present at the mitochondria but a fraction also localizes in the cytoplasm. Our work to understand altFUS localization upon environmental stimuli is ongoing but mitochondria seems to be its preferred subcellular localization.

The discovery of altFUS demonstrates the dual-coding nature of the FUS gene. It is very unlikely that altFUS is an exception and we hope this article will spark discussions and collaborations to investigate the importance of altORFs in human diseases.

[1] S. Samandi, A.V. Roy, V. Delcourt, J.-F. Lucier, J. Gagnon, M.C. Beaudoin, B. Vanderperre, M.-A. Breton, J. Motard, J.-F. Jacques, M. Brunelle, I. Gagnon-Arsenault, I. Fournier, A. Ouangraoua, D.J. Hunting, A.A. Cohen, C.R. Landry, M.S. Scott, X. Roucou, Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins, ELife Sciences. 6 (2017) e27860. https://doi.org/10.7554/eLife.27860.

[2] E. Kovacs, P. Tompa, K. Liliom, L. Kalmar, Dual coding in alternative reading frames correlates with intrinsic protein disorder, PNAS. 107 (2010) 5429–5434. https://doi.org/10.1073/pnas.0907841107.

[3] R. Pancsa, P. Tompa, Coding Regions of Intrinsic Disorder Accommodate Parallel Functions, Trends in Biochemical Sciences. 41 (2016) 898–906. https://doi.org/10.1016/j.tibs.2016.08.009.

[4] D.E. Andreev, P.B.F. O’Connor, C. Fahey, E.M. Kenny, I.M. Terenin, S.E. Dmitriev, P. Cormican, D.W. Morris, I.N. Shatsky, P.V. Baranov, Translation of 5’ leaders is pervasive in genes resistant to eIF2 repression, Elife. 4 (2015) e03971. https://doi.org/10.7554/eLife.03971.

[5] D.E. Andreev, P.B.F. O’Connor, A.V. Zhdanov, R.I. Dmitriev, I.N. Shatsky, D.B. Papkovsky, P.V. Baranov, Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes, Genome Biol. 16 (2015) 90. https://doi.org/10.1186/s13059-015-0651-z.

[6] J. Chen, A.-D. Brunner, J.Z. Cogan, J.K. Nuñez, A.P. Fields, B. Adamson, D.N. Itzhak, J.Y. Li, M. Mann, M.D. Leonetti, J.S. Weissman, Pervasive functional translation of noncanonical human open reading frames, Science. 367 (2020) 1140–1146. https://doi.org/10.1126/science.aay0262.

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FUS gene is dual-coding with both proteins united in FUS-mediated toxicity

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