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PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

ABSTRACT: Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.

SUBMITTER: Rudich P

PROVIDER: S-EPMC7117740 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

Rudich Paige P Watkins Simon S Lamitina Todd T

PloS one 20200402 4

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the ...[more]

PMID: 32240172

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Project description:Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expansion of a polyglutamine tract within the ATXN1 gene. Normal alleles have been reported to range from 6 to 35 repeats, intermediate alleles from 36 to 38 repeats and fully penetrant pathogenic alleles have at least 39 repeats. This distribution was based on relatively few samples and the narrow intermediate range makes the accuracy of the repeat sizing crucial for interpreting and reporting diagnostic tests, which can vary between laboratories. Here, we examine the distribution of 6378 SCA1 chromosomes and identify a very late onset SCA1 family with a fully penetrant uninterrupted pathogenic allele containing 38 repeats. This finding supports the theory that polyQ toxicity is related to the increase of the length of the inherited tracts and not as previously hypothesized to the structural transition occurring above a specific threshold. In addition, the threshold of toxicity shifts to a shorter polyQ length with the increase of the lifespan in SCA1. Furthermore, we show that SCA1 intermediate alleles have a different behavior compared to the other polyglutamine disorders as they do not show reduced penetrance when uninterrupted. Therefore, the pathogenic mechanism in SCA1 is distinct from other cytosine-adenine-guanine (CAG) repeat disorders. Accurately sizing repeats is paramount in precision medicine and can be challenging particularly with borderline alleles. We examined plasmids containing cloned CAG repeat tracts alongside a triplet repeat primed polymerase chain reaction (TP PCR) CAG repeat ladder to improve accuracy in repeat sizing by fragment analysis. This method accurately sizes the repeats irrespective of repeat composition or length. We also improved the model for calculating repeat length from fragment analysis sizing by fragment analyzing 100 cloned repeats of known size. Therefore, we recommend these methods for accurately sizing repeat lengths and restriction enzyme digestion to identify interruptions for interpretation of a given allele's pathogenicity.

Project description:Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2-7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

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Dataset Information

PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

Publications

PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

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