Over the past decade, there have been scattered reports of mammalian cells’ own DNA being found in the cytoplasm, mainly in the context of disease and aging. While it is not unusual to find DNA from an invading organism there, the presence of an organism’s own genetic code—in the form of complementary DNA (cDNA) synthesized from an RNA template—has puzzled scientists. In each case, the cDNA has come from endogenous retrotransposons, known for their copy-and-paste mechanism that results in the insertion of new copies of themselves into the genome. This process typically takes place in the nucleus, so the cytoplasmic cDNA lacked an explanation.
After detecting cDNA of Alu, the most abundant retrotransposon in the human genome, in the cytoplasm of cells modeling a degenerative eye disease, University of Virginia ophthalmologist Jayakrishna Ambati and his colleagues decided to investigate its mysterious origin. Their results, reported February 1 in PNAS, reveal that human cells can actually synthesize cDNA of Alu in the cytoplasm.
This is “potentially a path-breaking piece of information,” says Haig Kazazian, a geneticist at Johns Hopkins University who was not involved in this study.
Kazazian has studied retrotransposons for decades, focusing especially on LINE-1 (L1). L1 encodes a protein with the ability both to reverse transcribe RNA to DNA and to cut nucleic acids for that DNA to be inserted. In the late 1990s, he and his colleagues showed that the copy-and-paste mechanism of this mobile element is coupled: the reverse transcription takes place at the site where the genomic DNA is cleaved. L1 performs this trick to amplify itself in the genome, and Alu uses L1 to do the same.
Ambati’s study shows that L1 is reverse transcribing Alu in the cytoplasm, independent of genomic insertion. Kazazian says the finding is provocative, and that the evidence supporting it looks pretty good.
With this actually being discovered, scientists like myself are now going back to look at some of the phenomena we couldn’t understand and also couldn’t explain.
—Kang Zhang, Macau University of Science and Technology
Initiating DNA synthesis without genomic DNA poses an intriguing question: How does it happen without all the usual equipment involved? Reverse transcriptases need a short nucleic acid sequence—a primer—bound to the RNA template molecule in order to start. Typically, it is a short strand of nuclear DNA at the insertion site that serves this purpose for Alu reverse transcription but in the cytoplasm such a sequence is missing.
By looking at Alu’s sequence and its RNA structure, the team hypothesized that the retrotransposon could undergo self-priming—that is, the molecule folds back on itself. Only one other RNA molecule, from a rodent gene, had been shown to self-prime, but not in the cytoplasm. The data from the latest study suggest that, indeed, Alu is capable of self-priming, supplying the sequence from which the reverse transcriptase can initiate the synthesis. “That really provided the mechanistic explanation for how this event can actually happen in the cytoplasm, where there’s apparently nothing to prime it,” says Ambati.
The link from cytoplasmic cDNA to disease
Ambati and colleagues discovered the cytoplasmic synthesis of Alu while studying atrophic age-related macular degeneration (AMD). A decade ago, they found that Alu RNA accumulation induces cell death in a layer of the eye known as the retinal pigment epithelium (RPE), and this phenomenon is linked to the untreatable condition. Since then, a series of discoveries has offered a more detailed cellular picture of the disease—for instance, that Alu ultimately triggers inflammation, a hallmark of many age-related complications. In this new study, the researchers showed that Alu cDNA synthesized in the cytoplasm—not the one inserted into the genome—is essential for the toxicity in atrophic AMD.
See “An Eyeful of RNA”
Because Alu is a retrotransposon that relies on a reverse transcriptase to increase its number of copies, Ambati and colleagues hypothesized that nucleoside reverse transcriptase inhibitors (NRTIs)—drugs currently used to treat retroviral infections—could block Alu accumulation. In 2014, the team tested this idea in human and mouse cells and found that these drugs indeed prevented RPE degeneration, but did so through their previously unknown anti-inflammatory properties. In their latest work, the team showed that NRTIs also inhibit cDNA synthesis in the cytoplasm of RPE cells, pointing to NRTIs’ potential to block AMD on two fronts, both by opposing inflammation and by intercepting transcription.
Because of the strong evidence of the effectiveness of NRTIs in preventing RPE degeneration in the lab, and because these drugs have been used clinically for decades, the researchers decided to plunge into the archives of four independent health insurance databases in the United States. They wanted to assess whether the use of NRTIs for other purposes could have had the unintended benefit of reducing the incidence of atrophic AMD. Their analysis, including nearly 35 million adults, revealed a reduced risk of nearly 40 percent for developing atrophic AMD in patients taking NRTIs.
Ultimately, a prospective, randomized trial is needed to see if the drugs reduce the progression of atrophic AMD, says Ambati, who is also a cofounder of Inflammasome Therapeutics, a company focused on developing therapies for degenerative diseases. As NRTIs are toxic, his team has developed modified versions, known as Kamuvudines, which are equally effective, but safer, in cell culture and animal models. The company will likely start running clinical trials with Kamuvudines this year, according to Ambati.
“This is a very promising direction,” says the University of Rochester’s Vera Gorbunova, who was not part of the research. She adds that NRTIs could be potentially used as a treatment for various diseases related to inflammation. She studies aging and, as part of a team with whom Ambati also collaborated, has observed cytoplasmic accumulation of L1 cDNA in aged mice cells. Her team has previously discussed the unknown mechanism leading to formation of cytoplasmic cDNA, and referred to reverse transcription in the cytoplasm as one potential explanation.
“When we published that paper we had to really fight with the dogma of reverse transcription mechanism that was proposed some time ago—that it takes place in the nucleus [and that] it uses genomic DNA, which it probably does when things go well,” she says. “But when things do not go well, [reverse transcription] could probably happen in the cytoplasm. I’m glad to see this evidence.”
Macau University of Science and Technology ophthalmologist Kang Zhang, who has previously collaborated with Ambati and reviewed his latest paper, says that the finding of cDNA being synthesized in the cytoplasm might also be relevant to other conditions, such as cancer, neurodegeneration, and cardiovascular disease. “There are so many things happening in the cytoplasm. With this actually being discovered, scientists like myself are now going back to look at some of the phenomena we couldn’t understand and also couldn’t explain.” It is quite exciting, he says. “This is a paradigm shift.”
S. Fukuda et al., “Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration,” PNAS, doi:10.1073/pnas.2022751118, 2021.