Does New Role for ALS-Linked Protein Help Explain Neurodegeneration?

As the cell processes gene transcripts into mature mRNAs, just a few specific nucleotides mark sites where exons are spliced in, and introns spliced out. What if, by chance, those nucleotide motifs turned up in the middle of an intron? The result is a “cryptic” exon—one that looks to the spliceosome like the real McCoy, but ought never be incorporated into mRNA (see image below). Now, scientists claim that the job of policing these exons wannabes falls to TDP-43, an RNA-binding protein linked to most cases of amyotrophic lateral sclerosis and many instances of frontotemporal dementia and Alzheimer’s disease. Writing in the August 7 Science, researchers led by Philip Wong at Johns Hopkins University School of Medicine in Baltimore report that in cells lacking TDP-43, cryptic exons make their way into mRNA, and cell death rapidly ensues. Fixing the splice defect saved the cells. “For the first time, we now really understand what TDP-43 does,” Wong said. “I think suppressing cryptic exons is its principal function.”

Extra Exons.

The spliceosome should link exons, cutting out the whole intron, but without TDP-43 cryptic exons are often spliced into mRNA.[Image courtesy of Philip Wong; Science/AAAS.]

Other scientists who spoke with Alzforum were excited about the finding. “Their study revealed a novel function of TDP-43 in splicing,” commented Fen-Biao Gao of the University of Massachusetts Medical School in Worcester. However, he and others said splicing is one of many roles carried out by the protein. “TDP-43 is involved in multiple processes such as RNA splicing, RNA transport, translation, and microRNA biogenesis,” Gao said (see Feb 2014 news; Feb 2012 news; Highley et al., 2014). Robert Baloh of the Cedars-Sinai Medical Center in Los Angeles also noted that recent studies found TDP-43 maintains transcriptome integrity in other ways, by limiting double-stranded RNAs and silencing expression of mobile genetic elements like transposons (see Feb 2013 news; Li et al., 2012). “[Suppressing cryptic exons] is a very cool new function of TDP-43,” Baloh said. “It is hard to say this is the function that is causing disease, but it is a great lead.”

Three loose consensus sequences label an exon for the spliceosome. The key features include an adenine upstream of the splice site, an AG at the 5’ end, and a GU at the 3’ end. Sometimes, Wong said, nucleotides will line up in that pro-splicing pattern by chance. Some researchers call these pseudoexons. Such accidental splice sites are quite common—one study reported 103 of them in a single gene (Sun and Chasin, 2000). Researchers are finding that these accidental exons may contribute to disease (reviewed by Dhir and Burratti, 2010). Splicing mistakes have been linked to neurodegeneration; a mutation that allows an intron to be spliced into the ALS gene SOD1 causes familial disease (Valdmanis et al., 2009).

Wong and colleagues uncovered cryptic exons never before described. They only make it into mRNA when TDP-43 is missing. These exons occur in otherwise intronic segments, are not conserved between species, and have TDP-43 binding sites nearby. Wong found 50 of them in mouse embryonic stem cells, and 41 in the human HeLa cancer cell line. However, those numbers only reflect genes expressed by those specific cell types. “There could be hundreds of TDP-43 suppressed cryptic exons that we have not discovered yet,” he said. He has a paper under review that details cryptic exons in neurons.

First author Jonathan Ling initially noticed cryptic exons when he sequenced mRNA from a mouse embryonic stem cell line that had a Cre-inducible TDP-43 knockout. These cells die within a few days of TDP-43 loss, but first, they start making transcripts containing cryptic exons. Some of these altered mRNAs will likely acquire stop codons, and Wong presumes the cell destroys them by nonsense-mediated decay. Others may get translated into proteins carrying potentially harmful segments. Each of the 50 fake exons Ling found in these mouse cells occurred near a UG-repeat-rich region—these are known to attract TDP-43.

Ling and Wong hypothesized that if they could re-repress those cryptic exons, they could rescue the TDP-43 knockout cells. They created a hybrid protein comprising the RNA-targeting domains of TDP-43 and the splice-halting segment from a well-characterized splicing repressor, RAVER1. When they transfected their knockout mouse cells with this hybrid, it eliminated the cryptic exons from mature mRNAs and supported normal cell growth and survival (see image below). Wong concluded that TDP-43’s normal function is to suppress this wayward splicing.

RAVER1 to the Rescue.

Cells lacking normal TDP-43, but stably transfected with a version containing the splicing repressor RAVER1 (green), grow normally.[Courtesy of Philip Wong; Science/AAAS.]

What about cryptic exons in people? Since they are not normally expressed and there is no evolutionary pressure to retain these cryptic splice sites, they are not conserved among species. To find human versions that are regulated by TDP-43 regulation, Ling sequenced RNA from HeLa cells after silencing TDP-43 expression by RNA interference. He uncovered mRNAs containing 41 cryptic exons. All had a nearby TDP-43 binding site, and none matched the cryptic exons seen in mouse cells. Among them was ATG4B, a protease that participates in autophagy, which Wong noted has been linked to neurodegeneration (see Jul 2015 news; Jul 2014 news; Jul 2010 news). Another was RANBP1, which is involved in nuclear import. Wong speculated loss of RANBP1 might make it harder for the cell to send mislocalized cytoplasmic TDP-43 back to the nucleus.

Next the researchers used PCR to look for these nonconserved cryptic exons in postmortem tissue from people with TDP-43 proteinopathy. They tested brain samples from seven people who had died of FTD, ALS, or ALS-FTD and who had TDP-43 pathology. All contained transcripts with the erroneous splice forms.

How might this particular TDP-43 function be related to disease? The authors propose that in the nucleus, the protein binds the introns of unprocessed mRNAs and suppresses any nearby cryptic exons. If TDP-43 vacates the nucleus, as it does in disease states, it leaves those RNAs unprotected and the spliceosome includes the useless cryptic exons. With the transcriptome and proteome so altered, neurons degenerate, they suggest. The authors are now comparing their lists of mouse and human genes affected by cryptic exons, looking for commonalities that might be involved in neurodegeneration.

The theory lines up with results from a few other labs. Researchers discovered that TDP-43 often attaches to long introns, and Wong’s ideas would explain its presence there (see Mar 2011 news). In addition, researchers have discovered that boosting nonsense-mediated decay protects cells and animal models from TDP-43 proteinopathy (see Jun 2015 news). Sami Barmada of the University of Michigan Medical School in Ann Arbor said Ling’s work provides another reason to think nonsense-mediated decay is important for neuronal health.

Gene Yeo of the University of California in San Diego, one of the scientists who originally profiled TDP-43 RNA binding sites, found the results not entirely surprising. TDP-43 was already understood to regulate splicing of known, annotated exons, he said, so it makes sense that it also manages cryptic ones. While he found the study intriguing, he wondered about its relevance to human disease. Ling’s assay for cryptic splicing in brain tissue was not quantitative, he noted, so it could not tell if those brains contained one or 100,000 mRNAs with the cryptic exons. In addition, he said researchers need to look in cell types more relevant to human disease, not just mouse embryonic stem cells and the transformed HeLa line.

Wong said such work is ongoing; he wants to repeat these experiments in neurons derived from human induced pluripotent stem cells or fibroblasts, starting with healthy donors but eventually looking at disease states, including people with AD. In addition, he wants to test a gene therapy in mice that have no TDP-43 in their forebrain neurons, giving them the RAVER1 hybrid in the hopes of rescuing their defective splicing.

Gao was intrigued by another of Wong’s future projects: using the abnormal proteins made by cryptic transcripts as biomarkers for TDP-43 pathology. Wong’s lab is already designing antibodies to unique peptides that would only occur if cryptic exons are spliced into mRNAs, which would indicate TDP-43 left the nucleus. “If some of this protein gets into cerebrospinal fluid and you could detect it long before terminal neurodegeneration, that would be very cool,” Gao said.

Reference:
Ling JP, Pletnikova O, Troncoso JC, Wong PC. NEURODEGENERATION. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5. [PubMed.]


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disease-als disease-ftd tdp-43 topic-preclinical
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