A GGGGCC hexanucleotide repeat expansion in intron 1 of the C9ORF72 gene causes familial amyotrophic lateral sclerosis and frontotemporal dementia. Aberrant translation of this expansion results in toxic poly-dipeptides that build up in the central nervous system. However, the precise mechanism behind this repeat-associated non-AUG (RAN) translation has been unclear. Now, researchers led by Aaron Gitler, Stanford University, California, offer some clues. In the July 29 Nature Neuroscience, they report that a small ribosomal subunit, RPS25, drives RAN translation of a GGGGCC expansion in yeast. Knocking down the subunit limits poly-dipeptide production and boosts yeast survival. Knocking down homologs in fruit flies and cultured human motor neurons reduces neurodegeneration and cell death, respectively. Because RPS25 doesn’t appear to be required for global RNA translation, the authors suggest it could be a therapeutic target for neurodegenerative diseases caused by nucleotide repeats.

  • A small ribosomal subunit protein helps translate nucleotide repeats into poly-dipeptides.
  • Knocking out RPS25 reduces dipeptide repeat proteins in yeast and human cells.
  • This reduces neurodegenerative phenotypes in flies and cultured human cells.

“Their data is quite promising,” said Shuying Sun, Johns Hopkins University School of Medicine, Baltimore. Sun said there has been much debate about whether the repeat RNA or the associated poly-dipeptide repeat proteins (DPRs) cause the disease-related toxicity, and this paper suggests that targeting DPR production could be beneficial. However, she stressed that testing in animal models will be needed to be sure this is a safe target that doesn’t affect normal RNA translation.

First author Shizuka Yamada and colleagues went looking for genes involved in RAN translation. After assembling a library of yeast mutants, each lacking one of 275 genes that code for parts of the translational machinery, they introduced a 66-repeat GGGGCC construct into each cell line. In yeast without RPS25A, which encodes a ribosomal 40S subunit protein, RAN translation crashed while normal translation was unaffected. RPS25A has been found to be important in translation that bypasses the cap normally found on the 5' end of mRNA, such as during infection by viral RNA, but it doesn’t appear to play a major role in the translation of host mRNA (Hertz et al., 2013). 

Because RAN translation of GGGGCC repeats occurs in all six reading frames, it produces five different poly-dipeptide repeat proteins: glycine–alanine (GA), glycine–arginine (GR), proline–alanine (PA), proline–arginine (PR), and glycine–proline (GP). Genetically deleting RPS25A halved the level of poly(GP) in yeast. Similarly, in a human cell line with a 66-repeat expansion in C9ORF72, CRISPR-induced knockout of RPS25 halved the amount of poly(GP) relative to CRISPR controls. Poly(GA) and poly(GR) were reduced, as well.

To test these findings in more clinically relevant patient cells, the group used siRNA to knock down RPS25 in induced pluripotent stem cells from ALS patients who carried a C9ORF72 repeat expansion. This felled poly(GP) levels by more than 75 percent. In cultured motor neurons derived from patients, antisense oligonucleotides targeting RPS25 halved poly(GP) levels, protected against glutamate toxicity, and nearly doubled the proportion of surviving cells after 20 days. In flies expressing a 36-repeat GGGGCC construct, using RNAi to knock down RSP25 reduced poly(GP) fivefold and the flies lived twice as long as did untreated controls.

RPS25 seems to promote RAN translation of other nucleotide repeat expansions in addition to GGGGCC. In HeLa cells expressing a CAG repeat in ataxin-2, RPS25 knockout markedly reduced RAN-translated poly-alanine (poly(A)) and poly-glutamine. HeLa cells with a CAG repeat in ATXN2 likewise had less poly(A). All told, the results place RPS25 as central to RAN translation.—Gwyneth Dickey Zakaib

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References

Paper Citations

  1. . Ribosomal protein S25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting. Mol Cell Biol. 2013 Mar;33(5):1016-26. Epub 2012 Dec 28 PubMed.

Further Reading

Papers

  1. . RAN translation-What makes it run?. Brain Res. 2016 Sep 15;1647:30-42. Epub 2016 Apr 6 PubMed.
  2. . New developments in RAN translation: insights from multiple diseases. Curr Opin Genet Dev. 2017 Jun;44:125-134. Epub 2017 Mar 30 PubMed.
  3. . RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response. Nat Commun. 2017 Dec 8;8(1):2005. PubMed.
  4. . New pathologic mechanisms in nucleotide repeat expansion disorders. Neurobiol Dis. 2019 Oct;130:104515. Epub 2019 Jun 21 PubMed.

Primary Papers

  1. . RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats. Nat Neurosci. 2019 Jul 29; PubMed.