CAG Repeat Expansions Create Splicing Acceptor Sites and Produce Aberrant Repeat-containing RNAs

Overview

Journal bioRxiv

Date 2023 Oct 31

PMID 37904984

Authors

Rachel Anderson

Michael Das

Yeonji Chang

Kelsey Farenhem

Ankur Jain

Affiliations

Soon will be listed here.

Abstract

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames, without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs accounts for proteins that are attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

References

Sathasivam K, Neueder A, Gipson T, Landles C, Benjamin A, Bondulich M . Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci U S A. 2013; 110(6):2366-70. PMC: 3568346. DOI: 10.1073/pnas.1221891110. View

Eng L, Coutinho G, Nahas S, Yeo G, Tanouye R, Babaei M . Nonclassical splicing mutations in the coding and noncoding regions of the ATM Gene: maximum entropy estimates of splice junction strengths. Hum Mutat. 2003; 23(1):67-76. DOI: 10.1002/humu.10295. View

Yeo G, Burge C . Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol. 2004; 11(2-3):377-94. DOI: 10.1089/1066527041410418. View

Mouro Pinto R, Dragileva E, Kirby A, Lloret A, Lopez E, St Claire J . Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches. PLoS Genet. 2013; 9(10):e1003930. PMC: 3814320. DOI: 10.1371/journal.pgen.1003930. View

Nguyen L, Cleary J, Ranum L . Repeat-Associated Non-ATG Translation: Molecular Mechanisms and Contribution to Neurological Disease. Annu Rev Neurosci. 2019; 42:227-247. PMC: 6687071. DOI: 10.1146/annurev-neuro-070918-050405. View

Dulk S, Rouskin S . Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells. J Vis Exp. 2022; (190). DOI: 10.3791/64820. View

Walsh P, Fildes N, Reynolds R . Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 1996; 24(14):2807-12. PMC: 146018. DOI: 10.1093/nar/24.14.2807. View

Habara Y, Takeshima Y, Awano H, Okizuka Y, Zhang Z, Saiki K . In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene. J Med Genet. 2008; 46(8):542-7. DOI: 10.1136/jmg.2008.061259. View

Cheng W, Wang S, Mestre A, Fu C, Makarem A, Xian F . C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2α phosphorylation. Nat Commun. 2018; 9(1):51. PMC: 5754368. DOI: 10.1038/s41467-017-02495-z. View

10.

Labun K, Montague T, Krause M, Torres Cleuren Y, Tjeldnes H, Valen E . CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 2019; 47(W1):W171-W174. PMC: 6602426. DOI: 10.1093/nar/gkz365. View

11.

. Identification of Genetic Factors that Modify Clinical Onset of Huntington's Disease. Cell. 2015; 162(3):516-26. PMC: 4524551. DOI: 10.1016/j.cell.2015.07.003. View

12.

Paulson H . Repeat expansion diseases. Handb Clin Neurol. 2018; 147:105-123. PMC: 6485936. DOI: 10.1016/B978-0-444-63233-3.00009-9. View

13.

Gorbunova V, Seluanov A, Dion V, Sandor Z, Meservy J, Wilson J . Selectable system for monitoring the instability of CTG/CAG triplet repeats in mammalian cells. Mol Cell Biol. 2003; 23(13):4485-93. PMC: 164839. DOI: 10.1128/MCB.23.13.4485-4493.2003. View

14.

Jain A, Vale R . RNA phase transitions in repeat expansion disorders. Nature. 2017; 546(7657):243-247. PMC: 5555642. DOI: 10.1038/nature22386. View

15.

Watase K, Barrett C, Miyazaki T, Ishiguro T, Ishikawa K, Hu Y . Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels. Proc Natl Acad Sci U S A. 2008; 105(33):11987-92. PMC: 2503926. DOI: 10.1073/pnas.0804350105. View

16.

de Mezer M, Wojciechowska M, Napierala M, Sobczak K, Krzyzosiak W . Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference. Nucleic Acids Res. 2011; 39(9):3852-63. PMC: 3089464. DOI: 10.1093/nar/gkq1323. View

17.

Zheng G, Qin Y, Clark W, Dai Q, Yi C, He C . Efficient and quantitative high-throughput tRNA sequencing. Nat Methods. 2015; 12(9):835-837. PMC: 4624326. DOI: 10.1038/nmeth.3478. View

18.

Liao Y, Smyth G, Shi W . featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2013; 30(7):923-30. DOI: 10.1093/bioinformatics/btt656. View

19.

Soragni E, Petrosyan L, Rinkoski T, Wieben E, Baratz K, Fautsch M . Repeat-Associated Non-ATG (RAN) Translation in Fuchs' Endothelial Corneal Dystrophy. Invest Ophthalmol Vis Sci. 2018; 59(5):1888-1896. PMC: 5886103. DOI: 10.1167/iovs.17-23265. View

20.

Das M, Chang Y, Anderson R, Saunders R, Zhang N, Tomberlin C . Repeat-associated non-AUG translation induces cytoplasmic aggregation of CAG repeat-containing RNAs. Proc Natl Acad Sci U S A. 2023; 120(3):e2215071120. PMC: 9934169. DOI: 10.1073/pnas.2215071120. View