Pathogenic mutations within coding regions almost always change protein function. For a missense mutation in the DJ-1 gene that causes Parkinson’s disease, an entirely different mechanism may be at play. According to a study led by led by Rejko Krüger, University of Luxembourg, the G192C point mutation derailed interactions with the ribonucleoprotein U1, an essential component of the gene-splicing machinery, causing this apparatus to skip over the third exon during transcription. The stunted mRNA failed to translate into protein. A drug cocktail that strengthens U1 binding to DNA partly restored splicing in patient-derived neurons, and this corrected mitochondrial defects and spared dopaminergic neurons in midbrain organoid cultures. The findings, published in Science Translational Medicine on September 9, could have implications beyond this rare DJ-1 variant. The researchers report mutations at splice sites of other genes in people who have sporadic PD.

  • Point mutation in DJ-1 gene derails splicing machinery.
  • Transcripts lack exon 3 and make no protein.
  • Fixing the splicing defect restores DJ1 production and mitochondrial function in patient neurons.

Proper splicing is a critical part of gene expression, and the cell commits significant resources to the job. The spliceosome—a massive molecular machine consisting of several small nuclear RNAs that cooperate with as many as 300 proteins throughout the splicing process—coordinates the removal of introns and stringing together of exons. Mutations that interfere with docking the splicing machinery to heteronuclear RNA can lead to errors, such as exon skipping. Bungled splicing explains the pathogenicity of many mutations that cause inherited disease, even some that alter protein code (Xiong et al., 2015; Soemedi et al., 2017). Variants in several PD genes, including PINK1, PRKN, GBA, and a noncoding mutation in PARK7, which encoded DJ-1, may interfere with splicing, and in some cases, cause exon skipping (Samaranch et al., 2010; Asselta et al., 2014; Ghazavi et al., 2011). 

Exon Exclusion. In the normal PARK7 gene (top), the U1 small nuclear ribonucleoprotein (snRNP) base pairs with the splice donor site at the end of exon 3. The G>C mutation at the end of exon 3 disrupts this interaction, leading to exon skipping (second row). Mutating the U1 small nuclear RNA sequence (pink) to restore base pairing rescues splicing (bottom). [Courtesy of Boussaad et al., Science Translational Medicine, 2020.]

The 192G>C point mutation in the PARK7 gene encoding DJ-1 was previously described as a missense mutation that replaces a glutamic acid with an aspartic acid at position 64 of the protein (Hering et al., 2004). Later, when fibroblasts from mutation carriers became available, co-first authors Ibrahim Boussaad and Carolin Obermaier studied the mutation further. Something didn’t quite add up. Rather than finding a mutated protein, the researchers detected almost no DJ-1 in fibroblasts from two homozygous mutation carriers. In fibroblasts from two healthy heterozygous carriers, they detected roughly half as much protein as in cells from controls. DJ-1 was also largely absent from iPSC-derived neurons generated from the mutation carriers.

How did the mutation snuff out production of the protein? Stepped-up degradation was unlikely to blame, because neither autophagy inhibitors nor proteasome blockers nudged up DJ-1 protein levels. The researchers next looked to mRNA. While controls had a full-length 373 bp transcript, homozygous carriers had a stunted, 271 bp transcript. Heterozygous carriers expressed one full-length and one stubby transcript. Ultimately, the researchers discovered that exon 3 was missing from the latter (see diagram above).

The PARK7 point mutation occurs at the end of the shunned exon, right where the U1 snRNA base pairs with the transcript. Reasoning that the mutation disrupted this pairing, the researchers mutated the sequence of the U1 snRNA to restore complementarity. Sure enough, in iPSC-derived neurons from mutation carriers, transfection of the mutated U1 snRNA edged up DJ-1 protein, albeit only to 10 percent of the level of controls. Krüger believe that because the patient neurons still express normal U1 snRNA, it likely competes with the mutant copy and prevents a full rescue of splicing.

If the splicing defect produced a stunted mRNA transcript, why didn’t this transcript translate into a truncated protein, missing the 34 amino acids encoded by exon 3? Using a series of in vitro experiments, the researchers found that the stunted transcript was not translated. Computational modeling of RNA folding suggested that the secondary structure of the RNA rebuffed the translation machinery. This explains why only a smidgeon of DJ-1 protein was detected in neurons from mutation carriers.

The researchers next attempted a pharmacological approach to correct the splicing defect. They used a compound developed to treat another exon-skipping disease. Familial dysautonomia (FD) is a form of neurodegneration caused by a mutation in the IKBKAP gene that disrupts U1 snRNA binding (Slaugenhaupt et al., 2001). A screen identified a small molecule, subsequently dubbed RECTAS (rectifier of aberrant splicing) that corrected the splicing defect (Yoshida et al., 2015; Swanson, 2015). 

By itself, RECTAS did not trigger a reliable uptick in DJ-1 protein levels in the PD patient-derived cells. However, when the researchers combined RECTAS with phenylbutyric acid (PB), a compound previously reported to selectively boost PARK7 mRNA expression, DJ-1 protein levels inched up in fibroblasts of mutation carriers (Zhou et al., 2011). The small uptick in protein was sufficient to restore mitochondrial membrane potential, which had flagged in the mutant cells relative to controls. Mitochondrial dysfunction is a common pathological hallmark of PD, and DJ-1 is known to protect cells from oxidative stress and to regulate mitochondrial function. In iPSC-derived neurons treated with RECTAS+PB for 14 days, DJ-1 levels rose from being nearly undetectable to 17 percent of controls. PB is being tested in combination with a tauroursodeoxycholic acid as a potential treatment for amyotrophic lateral sclerosis and Alzheimer’s disease (Sep 2020 news).

To try a more physiological model, the researchers grew midbrain organoid cultures. Starting with neural epithelial stem cells, the researchers used an established cocktail of growth factors to turn them into neurons and various glial types, and used CRISPR to introduce the DJ-1 mutation in a subset of the samples. They treated the organoids with drugs for 25 days. Untreated PARK7 G192C organoids established significantly fewer dopaminergic neurons than did wild-type organoids, though both had a similar number of total neurons. Treatment of the organoids with the RECTAS/PB cocktail prevented this dopaminergic deficit (see image below).

Splicing Support. Midbrain organoid cultures with normal DJ-1 (top) had more dopaminergic neurons (red) than cultures with the 192G>C mutation (bottom). Treatment with PB+RECTAS dose-dependently increased dopaminergic neurons. [Courtesy of Boussaad et al., Science Translational Medicine, 2020.]

Together, the findings suggest that partial correction of aberrant DJ-1 splicing was sufficient to support the survival of dopaminergic neurons, presumably by restoring mitochondrial function.

These findings may point to a potential therapy for carriers of not just this rare mutation. Whole-exome-sequencing data from the Parkinson’s Progression Markers Initiative (PPMI), which includes 372 cases and 161 controls, showed mutations in U1 binding sites to be nearly 40 percent more common among people with PD. In a larger cohort of exomes from the Parkinson’s Disease Genome Sequencing Consortium project, these splice-site mutations were about 4 percent more common among 2701 people with PD than among 5713 controls. Among genes expressed in the brain, the splice-site mutations were about 20 percent more common among PD cases than controls. Krüger is investigating the potential role of splicing defects in some of these other genes.

While the researchers only searched for mutations in U1 snRNA binding sites, the human genome contains many other sites that could alter splicing if mutated, so splicing defects might be even more common than the PPMI and PDGSC data suggests. Mis-splicing likely plays a part in other neurodegenerative diseases, as well, Krüger suggested. Mutations in the AD risk gene ABCA7 have been found to cause splicing errors, plus researchers also clocked an uptick in bungled splicing of genes expressed in the hippocampi of AD patients (DeRoeck et al., 2018; Han et al., 2019). Therapeutics aimed at fixing splicing problems could theoretically prove effective for a subset of mutation carriers for many diseases, Krüger said.

“The practice of plotting mutation to dysfunction to shared pathology has already led to broader testing of therapies against genetic targets a-synuclein and LRRK2,” said Todd Sherer, Michael J. Fox Foundation. “PPMI exists for these validation and supplementary studies, and we’re hopeful that this work can translate to a next generation of trials.”

Jean-Christophe Rochet of Purdue University in Indiana found the findings convincing. Rochet had previously studied the mutation, and was puzzled how it only mildly affected the function of recombinant DJ-1, which does not undergo splicing. With the availability of patient-derived iPSCs expressing the full pre-mRNA, the pathogenicity of the mutation makes more sense, he said.

“Since there is a potential therapeutic approach for correcting these splicing errors, aberrant splicing should be more widely considered when investigating pathogenic mutations,” he said.—Jessica Shugart


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News Citations

  1. Micro Nudge? Positive Phase 2 Results for ALS Combination Drug

Paper Citations

  1. . RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease. Science. 2015 Jan 9;347(6218):1254806. Epub 2014 Dec 18 PubMed.
  2. . Pathogenic variants that alter protein code often disrupt splicing. Nat Genet. 2017 Jun;49(6):848-855. Epub 2017 Apr 17 PubMed.
  3. . PINK1-linked parkinsonism is associated with Lewy body pathology. Brain. 2010 Apr;133(Pt 4):1128-42. Epub 2010 Mar 30 PubMed.
  4. . Glucocerebrosidase mutations in primary parkinsonism. Parkinsonism Relat Disord. 2014 Nov;20(11):1215-20. Epub 2014 Sep 9 PubMed.
  5. . PRKN, DJ-1, and PINK1 screening identifies novel splice site mutation in PRKN and two novel DJ-1 mutations. Mov Disord. 2011 Jan;26(1):80-9. Epub 2010 Nov 8 PubMed.
  6. . Novel homozygous p.E64D mutation in DJ1 in early onset Parkinson disease (PARK7). Hum Mutat. 2004 Oct;24(4):321-9. PubMed.
  7. . Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. Am J Hum Genet. 2001 Mar;68(3):598-605. Epub 2001 Jan 22 PubMed.
  8. . Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia. Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2764-9. Epub 2015 Feb 9 PubMed.
  9. . Rectifying RNA splicing errors in hereditary neurodegenerative disease. Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2637-8. Epub 2015 Feb 17 PubMed.
  10. . Phenylbutyrate up-regulates the DJ-1 protein and protects neurons in cell culture and in animal models of Parkinson disease. J Biol Chem. 2011 Apr 29;286(17):14941-51. Epub 2011 Mar 3 PubMed.
  11. . An intronic VNTR affects splicing of ABCA7 and increases risk of Alzheimer's disease. Acta Neuropathol. 2018 Jun;135(6):827-837. Epub 2018 Mar 27 PubMed.
  12. . Identification of exon skipping events associated with Alzheimer's disease in the human hippocampus. BMC Med Genomics. 2019 Jan 31;12(Suppl 1):13. PubMed.

Further Reading


  1. . Familial dysautonomia is caused by mutations of the IKAP gene. Am J Hum Genet. 2001 Mar;68(3):753-8. Epub 2001 Jan 22 PubMed.
  2. . Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells. Nucleic Acids Res. 2018 Sep 6;46(15):7938-7952. PubMed.

Primary Papers

  1. . A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson's disease. Sci Transl Med. 2020 Sep 9;12(560) PubMed.