Stressed-Out Cells Translate C9ORF72 Repeats, Unleash Toxic Peptides

RNA translation goes something like this: Ribosomes glom onto a cap at the 5′ end of a neatly spliced mRNA strand, scan along until they bump into a well-positioned AUG, then start spooling off protein. But rules are there to be broken. Despite being buried in an intron and having no start codon, hexanucleotide expansions in the C9ORF72 gene are somehow translated into the dipeptide repeats (DPRs) implicated in ALS/FTD. Now, a trio of papers in Nature Communications proposes mechanisms for this so-called RAN (repeat-associated non-AUG) translation. Though their authors came to somewhat different conclusions about the particulars of the process, cell stress emerged as a common trigger for this atypical mode of protein synthesis. In particular, phosphorylation of the major translation initiation factor, eIF2α, turned on translation of DPRs, much as it does stress response genes.

C9orf72 Dipeptide Repeat Proteins. [Courtesy of Gendron et al., 2013. Acta Neuropathologica. CC BY 4.0 License.]

One of the studies, led by Clotilde Lagier-Tourenne of Massachusetts General Hospital in Boston, reported that translation of expanded C9ORF72 starts out with the ribosome finding the 5′ cap and subsequently scanning for AUGs. Things get a little wonky after that, as translation commences at a CUG codon, and ribosomes skip from one reading frame to another to produce three different DPRs. Another study led by Peter Todd of the University of Michigan in Ann Arbor came to similar conclusions regarding the cap-dependency of the process. He added that the cellular stress response promoted initiation at the CUG and potentially other non-AUG codons, and that the dipeptides, in turn, also ramped up the stress response. In a third paper, Shuying Sun and colleagues at Johns Hopkins University in Baltimore also implicated stress in the translation of DPRs; however, they found that DPRs could be translated from an excised intron lacking a 5′ cap.

“Despite some major discrepancies, the current papers have unraveled important aspects of the process [of RAN translation] in the context of C9ORF72,” wrote Ludo Van Den Bosch and Bart Swinnen of KU Leuven in a joint comment to Alzforum.

Hexanucleotide expansions within the first intron of the C9ORF72 gene are the most common genetic cause of ALS/FTD, where anywhere from 30 to thousands of copies of the GGGGCC sequence can wreak havoc on neurons. The field was shocked to learn that despite their location within the supposedly noncoding intron 1 of the gene, the repeats were in fact being translated into aggregation-prone dipeptide repeats (DPRs) (Feb 2013 news). What’s more, the DPRs were translated from three different reading frames, and from both the sense and antisense strands, making for a total of five different polypeptides: glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA), and glycine-proline (GP). RAN translation was evoked to explain DPR production, as it does for repeat peptides in other neurodegenerative diseases, including Huntington’s and spinocerebellar ataxias (Nov 2015 conference news). Exactly how this noncanonical translation happens, how it is regulated, and whether it might serve as a therapeutic target are all focuses of intense research.

A Nucleotide Away from Normal

Lagier-Tourenne’s study, published January 11, investigated how translation can occur in multiple reading frames. First author Ricardos Tabet and colleagues used DNA constructs containing 66 G4C2 repeats, which previously had been shown to produce peptides from three forward reading frames in cultured cells and in transgenic mice (Nov 2013 news on Gendron et al., 2013, and May 2015 news). Inserted into an expression vector, the 66 repeats were preceded by 113 nucleotides from the human C9ORF72 intron 1, which included no AUG start codon. To compare the efficiency of translation in the three forward reading frames from this single construct, the researchers inserted a protein tag—HA, His, or FLAG—downstream of the repeats in each reading frame. In rabbit reticulocyte lysate, a commonly used cell-free translation system, the researchers found that translation of poly-GA (in the +1 reading frame) was far more efficient than poly-GP (+2) and poly-GR (+3). Using uncapped mRNA decreased the translation of poly-GA fivefold, while poly-GP and poly-GR became undetectable.

Atypical Translation. In canonical translation (a), ribosomes, tRNA-Met, and other proteins scan mRNA until they encounter the AUG start codon. The same occurs in RAN translation (b), except it initiates at a CUG instead. Subsequent frameshifting may produce proteins products from other reading frames. [Courtesy of Tabet et al., 2018, Nature Communications, 2018.]

Using a series of mutated constructs, the researchers found that translation of all three peptides, regardless of reading frame, started at the same CUG codon located 24 nucleotides upstream of the repeat expansion. Translation depended on canonical translation machinery, including a cadre of cap-binding proteins and an initiator tRNA loaded with methionine, and the cap itself. Tabet determined that following initiation at this CUG, the translating ribosomes frameshifted, hopping over a nucleotide or two to generate peptides in the +2 and +3 reading frames. Ribosomal frameshifting can be induced by structural anomalies in mRNA, including the G-quadruplexes formed by these hexanucleotide expansions (Nov 2013 conference news).

While these experiments were not designed to detect translation of the reverse strand, Lagier-Tourenne said that antisense mRNA strands can be capped, too, so it is possible their translation is subject to similar rules. However, she noted that in human samples, antisense DPRs are exceedingly rare.

Stress Makes Things Less Stringent
As reported already December 8, Todd and colleagues also compared the efficiency of RAN translation in different reading frames. Using reticulocyte lysates and cellular models, they found, much as Tabet and colleagues had, that the ribosome more efficiently translated poly-GA than it did poly-GP, which in turn edged out production of poly-GR. PolyGA lies in-frame with the CUG codon. Only by mutating this CUG was Green able to shift expression of poly-GA in favor of poly-GP. Hence, Todd thinks that frameshifting does not fully explain translation of the three DPRs. Instead, he believes the ribosome may occasionally skip over the CUG for some reason, and then initiate translation at another codon in a different reading frame. However, he acknowledged to Alzforum that the DNA constructs he used were not designed to detect ribosomal frameshifting, and that it could play a role. In agreement with Lagier-Tourenne, Todd and colleagues reported that translation initiation was strongly cap-dependent.

Green and colleagues next set out to understand how cellular stress affected this cap-dependent, non-AUG translation. Previous studies have reported that the integrated stress response (ISR), which switches on in reaction to accumulating misfolded proteins, viral infection, and other insults, affects the stringency and kinetics of translation initiation. Specifically, the ISR triggers the phosphorylation of elongation initiation factor 2α (eIF2α), which favors noncanonical translation mechanisms, including cap-independent translation and non-AUG starts. Using stressors, eIF2α phosphorylation inhibitors, and other molecular tricks, the researchers found that stress-induced eIF2α phosphorylation suppressed global translation in HEK293 cells and in primary rat cortical neurons. However, these treatments had the opposite effect on expression of the DPRs, boosting expression of poly-GA, GP, and GR peptides in the cells. The same was true when the researchers analyzed translation of CGG repeats associated with Fragile X-associated tremor/ataxia syndrome.

Feed-Forward Frenzy.
The integrated stress response inhibits global translation but promotes RAN translation, which promotes stress granule formation and reduces global translation. The ISR also directly boosts granules and saps translation. [Courtesy of Green et al., 2017, Nature Communications.]

Finally, Green and colleagues uncovered a connection between RAN translation and stress granules. These membraneless organelles have been implicated in multiple neurodegenerative diseases, and may serve as a hotbed for protein aggregation (Oct 2015 newsOct 2016 news; May 2017 news). The DPRs boosted eIF2α phosphorylation and triggered the formation of stress granules. In sum, the researchers proposed a model in which cellular stress promotes toxic RAN translation, unleashing DPRs that induce the formation of stress granules and reduce global translation, thus implicating them in a feed-forward loop that promotes neurodegeneration.

“The stimulation of DPR translation by the integrated stress response is an exciting finding, and the induction of a feed-forward response in which both the stress response and DPR translation induce each other may prove to be an important step in the pathogenesis of C9ORF72-mediated disease,” commented Brian Freibaum of St. Jude Children’s Research Hospital in Memphis, Tennessee.

Caps Off to C9ORF72!
In the third paper, published January 4, researchers led by Sun also finger stress-driven eIF2α phosphorylation in RAN translation, but add a surprising twist. First author Weiwei Cheng and colleagues designed bicistronic constructs that allowed them to compare the efficiency of cap-dependent versus cap-independent translation of the repeats, as expressed in HeLa cells. Essentially, they found that cap-independent translation did occur, albeit 20–30 percent less efficiently than cap-dependent translation. They discovered that eIF2α phosphorylation induced by the ISR boosted cap-independent translation; this could be triggered by cytoplasmic accumulation of another protein linked to ALS/FTD—none other than TDP-43.

Cheng and colleagues wondered whether translation of the repeats occurred following excision of intron 1 of C9ORF72. Notably, introns lack 5′ caps and form a lariat structure when spliced out of RNA; this typically triggers their degradation in the nucleus. However, it appears that these RNA clippings sometimes make it out into the cytoplasm. Indeed, when the researchers expressed constructs containing the repeat-bearing intron flanked by the first two exons of C9ORF72, they found that not only did splicing occur, but the intron made its way into the cytoplasm, where it hooked up with ribosomes that made poly-GA and poly-GP. Again, eIF2α phosphorylation enhanced this translation.

Toxic Trimmings.
Dipeptide repeats are translated from a cap-less intron following its excision. Stress and eIF2α phosphorylation promotes this form of RAN translation. [Courtesy of Cheng et al., 2018, Nature Communications.]

Sun proposed that the structure of the excised, repeat-containing intron somehow facilitates cap-independent translation. She pointed out that although ribosomes translate capped transcripts more efficiently than uncapped, excision of C9ORF72 intron 1 is far more common than its aberrant retention, and thus the excised intron may be the most common C9ORF72 repeat substrate available for translation in the brain. She plans to investigate how the excised intron manages to escape degradation in the nucleus and gain entry into the cytoplasm for translation.

Lagier-Tourenne, on the other hand, thinks efficiency of translation is the most important factor, and therefore cap-dependent translation predominates (Niblock et al., 2016). However, when she tested this in the cell-free system, she found yet another twist. An upstream open reading frame with two consecutive stop codons in intron 1, just before the expansion, snagged scanning ribosomes before they could reach the repeats, nearly abolishing RAN translation. She told Alzforum that this does not necessarily imply that the excised intron hypothesis trumps the cap-dependent translation idea, because small upstream ORFs are established regulatory motifs. Cellular stress, which inhibits AUG-initiated translation, and even neuronal activation, might suppress translation of the uORF, enabling RAN, she said.

Todd thinks all of these mechanisms could happen in cells to some extent. “There is a lot of noise in nature, and this is especially true of translation,” he said. Lagier-Tourenne agreed, noting that because the studies each used different DNA constructs and expression systems, a direct comparison is difficult. Todd noted that despite different conclusions on cap dependence, both his and Sun’s studies converged on the role of the stress response in promoting RAN translation. “Stress both weakens codon fidelity, and enhances cap-independent translation,” he said.

Van Den Bosch and Swinnen also focused on this convergence. “The observation that stress induces a feed-forward loop enhancing RAN translation is extremely interesting and fits in a broader perspective,” they wrote. “It suggests a self-sustaining mechanism in which stress induced by either arginine-containing DPRs (PR and GR), or by the repeat RNA itself, fuels a vicious circle,” they added (Swinnen et al., 2018).

Todd believes something as essential as translation likely makes a poor therapeutic target. “But intervening in the stress response could have therapeutic legs,” he said. Indeed, Sun reported that small-molecule inhibitors of PRKR-like ER kinase (PERK), which phosphorylates eIF2α in response to accumulating unfolded proteins, substantially blocked RAN translation. These compounds have been tested in animal models of neurodegenerative disease, including ALS/FTD, but turned out to be toxic (Oct 2013 news). Researchers have begun screening for less-toxic leads (May 2017 news; Halliday et al., 2017). High levels of activated PERK and p-eIF2α have been reported in AD and PD brains, as well (Hoozemans et al., 2007Hoozemans et al., 2009).

Trying another approach, Lagier-Tourenne and colleagues took a hint from the uORF that suppresses RAN, generating a series of antisense oligonucleotides (ASOs) to adhere to the mRNA upstream from the repeats, to block scanning ribosomes. These constructs hindered RAN from transcripts with no uORF. The researchers proposed that blocking ribosomal scanning in this way could serve as a therapeutic strategy to prevent translation of the DPRs. ASOs that suppress proteins with repeat expansions reduce toxicity in animal models of ataxia and ALS (May 2017 news). In people with Huntington’s disease, this strategy curbs production of expanded huntingtin, and nusinersen, an ASO that corrects aberrant splicing of the spinomuscular atrophy gene, was approved by the U.S. Food and Drug Administration in 2016 (Dec 2017 newsNov 2016 news).

All three study leaders emphasized that it will be crucial to examine how RAN translation actually occurs in neurons from C9ORF72 repeat expansion carriers. Lagier-Tourenne proposed monitoring translation of the DPRs after using CRISPR to mutate the putative CUG start codon in induced pluripotent stem cell (iPSC)-derived neurons from patients. Freibaum added that it would be interesting to visualize the 5′ cap on the G4C2 transcript in such cells, while Sun aims to investigate the transport and translation of the excised intron.

Featured Papers

Tabet R, Schaeffer L, Freyermuth F, Jambeau M, Workman M, Lee CZ, Lin CC, Jiang J, Jansen-West K, Abou-Hamdan H, Désaubry L, Gendron T, Petrucelli L, Martin F, Lagier-Tourenne C. CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts. Nat Commun. 2018 Jan 11;9(1):152.  PubMed.

Green KM, Glineburg MR, Kearse MG, Flores BN, Linsalata AE, Fedak SJ, Goldstrohm AC, Barmada SJ, Todd PK. RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response. Nat Commun. 2017 Dec 8;8(1):2005. PubMed.

Cheng W, Wang S, Mestre AA, Fu C, Makarem A, Xian F, Hayes LR, Lopez-Gonzalez R, Drenner K, Jiang J, Cleveland DW, Sun S. C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2α phosphorylation. Nat Commun. 2018 Jan 4;9(1):51. PubMed.


Gendron TF, Bieniek KF, Zhang YJ, Jansen-West K, Ash PE, Caulfield T, Daughrity L, Dunmore JH, Castanedes-Casey M, Chew J, Cosio DM, van Blitterswijk M, Lee WC, Rademakers R, Boylan KB, Dickson DW, Petrucelli L. Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol. 2013 Oct 16; PubMed.
Niblock M, Smith BN, Lee YB, Sardone V, Topp S, Troakes C, Al-Sarraj S, Leblond CS, Dion PA, Rouleau GA, Shaw CE, Gallo JM. Retention of hexanucleotide repeat-containing intron in C9orf72 mRNA: implications for the pathogenesis of ALS/FTD. Acta Neuropathol Commun. 2016 Feb 25;4:18. PubMed.

Swinnen B, Bento-Abreu A, Gendron TF, Boeynaems S, Bogaert E, Nuyts R, Timmers M, Scheveneels W, Hersmus N, Wang J, Mizielinska S, Isaacs AM, Petrucelli L, Lemmens R, Van Damme P, Van Den Bosch L, Robberecht W. A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism. Acta Neuropathol. 2018 Jan 4; PubMed.

Halliday M, Radford H, Zents KA, Molloy C, Moreno JA, Verity NC, Smith E, Ortori CA, Barrett DA, Bushell M, Mallucci GR. Repurposed drugs targeting eIF2α-P-mediated translational repression prevent neurodegeneration in mice. Brain. 2017 Jun 1;140(6):1768-1783. PubMed.

Hoozemans JJ, van Haastert ES, Eikelenboom P, de Vos RA, Rozemuller JM, Scheper W. Activation of the unfolded protein response in Parkinson’s disease. Biochem Biophys Res Commun. 2007 Mar 16;354(3):707-11. Epub 2007 Jan 17 PubMed.

Hoozemans JJ, van Haastert ES, Nijholt DA, Rozemuller AJ, Eikelenboom P, Scheper W. The unfolded protein response is activated in pretangle neurons in Alzheimer’s disease hippocampus. Am J Pathol. 2009 Apr;174(4):1241-51. PubMed.

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c9orf72 dipeptide repeat proteins disease-als disease-ftd DRPs eIF2α PERK stress response topic-preclinical
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