NEW YORK (GenomeWeb) – Although Drosha is widely known as a key player in microRNA processing, new data out of Mount Sinai Hospital indicating that the RNA nuclease also serves to fight virus infection in mammalian cells, cleaving viral RNA during infection and altering the cellular environment to block replication.
The findings suggest that Drosha may be used by mammalian cells for a task other than that which it evolved to do, and the Mount Sinai team suggests in its study that this repurposing has turned an miRNA-related nuclease into a cellular defender.
But the senior author of the report has also proposed that it might, in fact, be the other way around, with Drosha starting off as an antiviral factor but later developing into a component of the miRNA machinery.
The RNAi pathway is well established as an antiviral mechanism in plants, nematodes, and arthropods, which produce virus-derived small interfering RNAs in response to infection. Mammalian cells, however, rely on the protein-based type I interferon (IFN-I) response to fight off virus infection, and it is widely believed that they lack any antiviral RNAi mechanism.
What mammals do have is a microRNA system, which involves much of the same cellular machinery as RNAi including the RNase III enzymes Drosha (in the nucleus) and Dicer (in the cytoplasm), which process the non-coding RNAs into small RNAs that induce target gene silencing.
But there have been recent reports of antiviral RNAi in certain mammalian cells, namely kidney cells and stem cells, although this topic remains controversial. At the same time, it has been demonstrated by a number of different groups that miRNAs can be used to block viral replication by engineering viruses to contain sequences of perfect complementarity to an miRNA of choice, which indicates that viruses have not evolved any way to combat miRNA-based attacks.
To Mount Sinai's Benjamin tenOever, such findings raised the questions about why RNA viruses, with a few notable exceptions such as herpes viruses and hepatitis C, don't take advantage of a host's miRNA system as part of their replication strategy.
"Your average, run-of-the-mill virus does not produce small RNAs," he told Gene Silencing News. "It was largely assumed it doesn't happen because they can't do it."
To answer the question, tenOever and his team began incorporating miRNA precursors into different viruses to see if miRNAs would be produced and, if they did, would they silence their targets.
"We entirely expected the answer to be, 'No,' especially for the cytoplasmic viruses because the components necessary to start that processing mechanism are all in the nucleus," he said, referring to Drosha. "In reality, the answer was, 'Yes, they can make copious amounts of microRNAs.' If you put the most basic precursor of a microRNA into any virus, regardless of the polarity of the genome or where it replicates … it will generate functional small RNAs that are processed perfectly."
As they reported in RNA, the scientists found that Drosha can be relocalized into the cytoplasm upon viral infection and they speculated that it may represent "a vestigial remnant of the small RNA-mediated defense mechanism that has been evolutionarily retained from plants, worms, and flies and repurposed in the cleavage of mRNA during times of cellular stress."
In a paper appearing this week in the Proceedings of the National Academy of Sciences, the investigators described the follow up to this work.
They started by screening a panel of diverse viruses and pathogen-associated molecular patterns, which were found to all be capable of inducing the relocalizing Drosha to the cytoplasm. They then examined the impact of Drosha and Dicer on the replication of a positive sense virus and negative sense virus, with Drosha being identified as a restriction factor in viral replication.
To better understand the mechanism for Drosha’s antiviral activity, they studied the biology of cytoplasmic Drosha, finding that depletion of the nuclear export protein CRM1 resulted in a loss of Drosha-dependent processing of virus-derived primary miRNAs.
"Furthermore, we implicated serine 300 and 302 in the virus-inducible translocation of Drosha and showed that cytoplasmic localization is required to confer the full antiviral activity of Drosha," they wrote in PNAS.
Lastly, they presented small RNA data from both mammalian and insect virus infections showing that loss of Drosha "did not impact the small RNA profile of specific viral RNAs, but, rather, loss of Drosha enhanced the presence of viral RNA, presumably as a result of increased replication.
"In addition, mRNA seq data from infected cells with or without Drosha demonstrated Drosha-dependent changes in the host transcriptome that, in concert with the cleavage of viral RNA, likely contribute to its antiviral property," they wrote.
Based on these results, tenOever and his team stated that Drosha appears to represent "a unique and conserved arm of the cellular defenses used to combat virus infection." But just how that came is a matter for debate.
In their paper, the Mount Sinai group suggests that Drosha is being repurposed from its job as an miRNA processor into a virus cleaver — a conclusion that tenOever said is straightforward enough.
"You've already got microRNAs, you've already got processing machinery — it would be easy to envision that … exporting your nuclease into the cytoplasm would be a great … antiviral defense since its essential activity is to cut up double-stranded RNA," he said.
However, when you consider the biology of Drosophila, this hypothesis "falls apart a little bit," he noted.
The apparent repurposing of Drosha occurs in these flies, but the animals are also capable of antiviral RNAi. "The idea of repurposing [a fruitfly's] microRNA components to contribute to its RNAi response is possible, but makes me think there might be more there," he said.
Instead, tenOever speculates that Drosha may have originated as an antiviral agent, predating microRNAs and the RNAi pathway. Only later did it become part of these gene-silencing mechanisms.
"That would explain why mammals don't have RNAi," he said. "Maybe we never had RNAi." Instead, it is possible that Drosha in arthropods and plants evolved into an RNAi, and mammals developed the adaptive immune responses necessary to deal with the variety of viruses they encounter while retaining still retaining Drosha-based defense.
He conceded, however, that such a theory is difficult to prove. Doing so would require expertise that he does not have and is perhaps best handled by "people more experienced in this evolutionary space."