RNA Metabolism in ND 2015: RNA Regulator Locked Out of Nucleus by C9ORF72 Repeats

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Cells possess a protein that can alleviate C9ORF72 repeat toxicity—ironically, it may not be able to reach the nuclear RNA repeats to silence them. Christian Haass of the German Center for Neurodegenerative Diseases (DZNE) in Munich has proposed that the RNA-binding protein hnRNP A3 dismantles the repetitive RNAs associated with some cases of amyotrophic lateral sclerosis and frontotemporal dementia. Heterogeneous nuclear ribonucleoprotein A3 must enter the nucleus to clear the repeat RNAs, but they, in turn, interfere with normal nucleocytoplasmic transport. HnRNP A3 represents the first reported victim of this defective nuclear entry, Haass told Alzforum. He presented his data at the RNA Metabolism in Neurological Disease meeting held October 15-16, a satellite of the Society for Neuroscience annual conference in Chicago.

Double trouble. HeLa cells accumulate polydipeptides (red) encoded by the C9ORF72 repeat as well as aggregated p62 (green), a common marker of ALS inclusions. [Courtesy of Christian Haass, DZNE]

The C9ORF72 gene, which encodes a protein of as-yet-undetermined function, normally contains up to 23 hexanucleotide repeats. That number can grow into the hundreds or thousands, and when it does, it amplifies risk for ALS and FTD. Scientists are not yet sure how the repeats cause disease, but the sequences are abnormally transcribed into RNAs that are translated into polydipeptide repeats (DPRs). These RNAs and DPRs aggregate into foci and inclusions, respectively. Scientists recently reported that either the RNAs or DPRs, or both, interfere with protein trafficking across the nuclear pore (see Aug 2015 news).

After C9ORF72 repeats were linked to neurodegeneration (see Sep 2011 news), Kohji Mori in Haass’ lab went hunting for RNA-binding proteins that might interact with the repeat RNA. He identified heterogeneous nuclear ribonucleoprotein (hnRNP) A3, an RNA transporter (see Feb 2013 news). This hnRNP possesses sequences similar to hnRNP A1 and hnRNP A2/B1, which are mutated in rare ALS cases and also bind C9ORF72 repeat RNAs (see Mar 2013 news). Scientists have not yet discovered hnRNP A3 mutations in ALS, but Haass predicts they will (Calini et al., 2013).

As Mori and Haass examined autopsy tissues from C9ORF72 repeat carriers, they noticed that hnRNP A3 was often absent from the nucleus, where it normally resides. It also turned up in aggregates in both the nucleus and cytoplasm, and occasionally co-localized with p62, a common component of pathological inclusions in ALS. The researchers suspected that since it was mislocalized, hnRNP A3 failed to perform its normal function in the C9ORF72 patients.

To test the effects of hnRNP A3 loss of function in the lab, Mori used small interfering RNAs to knock down hnRNP A3 in HeLa cells expressing 80 copies of the C9ORF72 hexanucleotide repeat. These cells generate repeat RNA foci and produce repeat polydipeptides, which sometimes aggregate with p62 (see image above). In the hnRNP knockdowns, the results were striking. The repeat RNA shot up to much higher levels. “It was a day-and-night difference,” Haass told Alzforum. Without hnRNP A3, the cells overproduced the repeat RNA by six- or sevenfold, he said, and accumulated many more RNA foci. The cells also made much more of the repeat polydipeptides than control cells did. Mori and collaborator Dieter Edbauer of DZNE obtained similar results when they knocked down hnRNP A3 in primary rat neurons.

If loss of hnRNP A3 allowed C9ORF72 repeats to run amok, then augmenting expression of the RNA-binding protein would tone that down, the authors predicted. Sure enough, overexpressing hnRNP A3 in HeLa cells diminished both repeat RNA and polydipeptide proteins.

How does hnRNP A3 work? Because C9ORF72 repeat RNA folds back on itself in tight hairpins, the researchers suspected hnRNP A3 altered this conformation. Haass that suggested hnRNP A3 binds to the repeats, unwinding their secondary structure and exposing them to degradation by RNases. Consistent with this, hnRNP A3 sans RNA-binding domain failed to attenuate C9ORF72 pathology in HeLa cells lacking the normal hnRNP A3 gene.

Back when they looked at human tissues, Mori and Haass had noticed that hnRNP A3 was missing from the nucleus, so they predicted it might be unable to protect cells from repetitive RNAs if it was mislocalized. To find out, Mori removed the nuclear localization signal from the hnRNP A3 gene and expressed it in their modified HeLa cells. Compared with the wild-type gene this construct weakly rescued cells from C9ORF72 pathology, reducing repeat RNAs and polydipeptides by about half. Haass attributed the partial rescue to some hnRNP A3 that made it into the nucleus by chance, which he said often occurs with nuclear proteins lacking their nuclear localization sequence.

Together, these findings led Mori and Haass to propose that hnRNP A3 represses expression of C9ORF72 repeats, but only if it can get into the nucleus. They predicted that less hnRNP A3 in the nucleus would spell more C9ORF72 repeat pathology. The scientists tested this in human autopsy tissues. So far, they have examined the brains of 18 people who died of C9ORF72-based ALS-FTD. Mori calculated the ratio of hnRNP A3 to poly-glycine-alanine inclusions in individual neurons. Indeed, the less hnRNP A3 he observed in the nucleus, the more polyGA pathology in the cell.

“This is interesting because they identified an RNA-binding protein that suppresses DPR protein production,” commented Fen-Biao Gao of the University of Massachusetts Medical School in Worcester, who co-organized the conference. Gao told Alzforum he found the results from human tissues to be particularly striking.

C9ORF72 repeats could instigate a vicious cycle, Haass theorized. Once a few escape the nucleus and interfere with nucleocytoplasmic transport, hnRNP A3 could get stuck out in the cytoplasm. It would be unable to unwind newly transcribed C9ORF72 RNA, allowing the concentration of repeat RNAs and dipeptides to skyrocket and further blocking entry of hnRNP A3, and other proteins, to the nucleus.

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