Proteins that can aggregate and cause neurodegeneration need to be tightly regulated, lest excessive production trigger disease. How, then, do cells hit the sweet spot for such gene expression? In the May 19 Nature, researchers led by Rohan de Silva at UCL Queen Square Institute of Neurology, London, propose a new mechanism based on research into the MAPT gene that encodes tau. They found that a natural antisense transcript, MAPT-AS1, contains a transposable element known as a mammalian-wide interspersed repeat (MIR). Short sequences within this MIR interfered with the binding of MAPT mRNA to ribosomes, thus blocking translation. In cultured cells, increasing MAPT-AS1 lowered tau protein, while silencing it boosted tau. In human brain, low MAPT-AS1 correlated with having more tau tangles, hinting at a link to pathology.
- A new type of antisense transcript controls production of tau.
- MIR-NATs interfere with ribosome function.
- They exist for many neurodegenerative disease genes.
The scope of this regulatory mechanism may extend beyond tau. The authors found similar MIR natural antisense transcripts (MIR-NATs) for several other genes tied to neurodegenerative disease, including α-synuclein and APP. Notably, many of those genes encode proteins with little secondary structure. These intrinsically disordered proteins are prone to aggregate and wreak havoc in the brain. De Silva believes the evidence hints at a conserved mechanism for keeping potentially harmful proteins in check. “This could represent a new class of gene regulation that is especially relevant to neurodegeneration,” he told Alzforum.
Others said the findings were important. “This is an eye-popping and elegant study,” noted Peter Nelson at the University of Kentucky, Lexington (full comment below). Veroniki Nikolaki, working in Fen-Biao Gao’s laboratory at the University of Massachusetts Medical School, was intrigued by the potential applications. “This is an exciting discovery, because it raises the possibility that boosting expression of this specific antisense RNA may be a novel therapeutic avenue,” she wrote to Alzforum (full comment below).
MIR Control? The MAPT gene (top) has three antisense MIR-NATs (bottom), each with slightly different exons (gray boxes). Their 5' termini complement MAPT mRNA, in one case overlapping the 5' UTR (blue), while their 3' ends contain an MIR element (red) that regulates translation. [Courtesy of Simone et al., Nature.]
Genetic studies have identified thousands of natural antisense transcripts in the human genome, but so far there have been few functional studies detailing their effects (for review, see Pelechano and Steinmetz, 2013; Statello et al., 2020). MAPT-AS1 was previously identified; its transcription begins in the 5' untranslated region (UTR) of the MAPT gene, but on the opposite DNA strand, and extends in the opposite direction. It complements only a small portion of the MAPT mRNA. Previously, researchers led by John Kwok, now at the University of Sydney, found unusually low amounts of MAPT-AS1 in Parkinson’s disease compared to control brain (Coupland et al., 2016).
To find out what this partial antisense transcript might be doing, first author Roberto Simone in de Silva’s group silenced MAPT-AS1 in a neuroblastoma cell line using small interfering RNA. The amount of tau protein in the cells tripled, while MAPT mRNA remained unchanged. Conversely, overexpressing MAPT-AS1 slashed tau protein by two-thirds, while again MAPT mRNA stayed stable. Notably, in cells with excess MAPT-AS1, fewer ribosomes attached to MAPT mRNA, confirming that the antisense transcript somehow blocked translation.
How did MAPT-AS1 do this? Simone and colleagues investigated its transcription, finding three different isoforms. The start of one, dubbed t-NAT1, began just inside the 5' UTR of the MAPT gene, while the other two, t-NAT2l and t-NAT2s, started even further along the MAPT sequence in a conserved intron (see image above). All three transcripts extended away from MAPT in the reverse direction, and all three shared the same 3' sequence, which contained a mammalian-wide interspersed repeat of 62 nucleotides. Deleting either the 5' tau-antisense region or the MIR element from MAPT-AS1 abolished its suppression of tau synthesis. On the other hand, a “miniNAT” consisting of only the 5' region and the MIR element retained full functionality, demonstrating that these two regions were necessary and sufficient for gene regulation.
Digging deeper, the authors found that the MIR element contained two seven-nucleotide sequences that were essential for function. Though these are upstream of the MAPT gene, one exactly repeated a seven-nucleotide block in the 5' region of MAPT, and the other exactly complemented a second MAPT heptanucleotide. MAPT mRNA needs these short sequences to bind 18S ribosomal RNA, part of the small ribosomal subunit, and initiate translation. The doppelganger heptanucleotide in the MIR element competed with MAPT mRNA for binding to ribosomal RNA, while the antisense motif bound directly to MAPT mRNA, blocking ribosome recruitment. Deleting either MIR motif abolished repression of tau synthesis.
Because these heptanucleotide motifs are common throughout the genome, being used by many different transcripts to snag the 18S ribosome, the authors believe the MIR versions could represent a general mechanism for interfering with ribosomal RNA binding. However, they can’t work alone. Altogether, the experiments suggested that while the MIR element mediates translational repression, the 5' region confers robustness and specificity, in this case for the MAPT gene.
Does this mechanism work in vivo? The authors used an adenoviral vector to express either full-length t-NAT1 or miniNAT in the hippocampi of mice carrying humanized tau genes (Andorfer et al., 2003). Eight weeks later, these mice had half as much tau protein as did controls. The relationship held in human brain as well. The authors examined data from the Religious Orders Study and Rush Memory and Aging Project cohort, and found an inverse relationship between Braak stage and the amount of MAPT-AS1 in 636 postmortem samples.
The data dovetail with Kwok’s findings from PD brain, where reduced MAPT-AS1 expression correlated with Parkinson’s disease and increased tau production, particularly the four-repeat isoforms of tau. Although Parkinson’s is not a classic tauopathy, tau deposits are present in this disorder (Sep 2016 news; Zhang et al., 2018).
The MIR-NAT mechanism may also help explain why some genetic variants increase the risk for tauopathies. The H1 haplotype of the MAPT gene boosts the risk for PD as well as two other tauopathies, progressive supranuclear palsy and corticobasal degeneration (Mar 2019 news). One of the risk SNPs in this haplotype, rs62056779, sits in the 5' untranslated region of MAPT and encourages binding to ribosomal RNA, which might be stopped by MAPT-AS1. Two more risk SNPs, rs17690326 and rs17763596, are found in the MIR element of MAPT-AS1, suggesting they may affect the antisense mechanism directly.
Susanne Wegmann at the German Center for Neurodegenerative Diseases in Berlin noted that regulation of tau production at the ribosome could help explain the protein’s distribution to different neuronal compartments. “It will be very interesting to see whether local tau translation is regulated by controlled release of MIRs at sites of translation, and if aberrant somatodendritic tau translation in response to Aβ involves repression of tau-targeted NATs,” she wrote to Alzforum (full comment below).
Common Regulation. Many proteins with associated MIR-NATs (black circles) are linked to neurodegenerative disease and connect with each other through protein interaction networks (green dots). [Courtesy of Simone et al., Nature.]
How common is this form of gene regulation? Simone searched for MIRs within known antisense transcripts in genomic data from GENCODE. He found nearly 1,200 MIR-NATs in the human genome, making up about 6 percent of all long noncoding RNAs. Almost half of these MIR-NATs overlapped the 5' region of a protein-coding gene, and many of these genes associated with neurological conditions such as dementia, Parkinson’s, amyotrophic lateral sclerosis, and neuropathy (see image above).
The authors investigated one of these antisense transcripts, PLCg1-AS, and found that it lowered the phospholipase C γ1 protein in cultured neuronal cells, as expected. PLCγ1 and the related protein PLCγ2 have been linked to Alzheimer’s disease (Jun 2020 news; Kim et al., 2021). There may be more AD genes controlled by MIR-NATs. Examining RNA-Seq datasets from AD brain, the authors found 446 MIR-NAT/gene pairs that were differentially expressed compared to control brain. Almost half of these genes encoded intrinsically disordered proteins, and many of the proteins associated with each other in protein interaction networks.
To Simone and de Silva, this suggests that MIR-NATs provide an extra layer of regulation for aggregation-prone proteins. They plan to further investigate MIR-NAT/gene pairs. Because mice do not have MIR elements, they will use human cell and organoid models.
De Silva is also interested in the therapeutic potential of MIR-NATs. He noted that they work differently than antisense oligonucleotides and microRNA, both of which form duplexes with target genes, leading to degradation of the transcripts. MIR-NATs directly block translation without affecting the amount of transcript, and so could be an alternative approach.
Kwok noted that the recent success of RNA-based COVID-19 vaccines demonstrates the feasibility of using RNA therapeutically. “What is needed now is a concerted effort to design strategies for delivering these NAT molecules into the brain in a safe manner and at therapeutically relevant levels,” he wrote to Alzforum (full comment below).—Madolyn Bowman Rogers
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