Collaboration between two PRBB groups dismantles a mistaken assumption about viruses

The group led by Juana Diez at the Department of Medicine and Life Sciences (MELIS-UPF) studies the biology of pathogenic viruses: how they enter cells and take over, or how they interact with the infected host.

Eva Novoa and her group at the Centre for Genomic Regulation (CRG) are experts in RNA modifications.

“For some years now, there has been a boom in RNA modifications and their key importance in many biological processes,” explains Novoa. “Currently, due to the Covid19 pandemic, among other things, there is another big boom in RNA viruses. So understanding RNA modifications in viruses is a topic of great interest right now,” explains Novoa.

This explains the collaboration of the two groups, and how they began to study these modifications – to discover, to their surprise, that the best-described modification did not, in fact, occur.

“Being in a place like the PRBB, where there are many laboratories with expertise in different subjects, has made this collaboration much easier for us”
Juana Díez (MELIS-UPF)

Rewriting the story

It has been known for about eight years that cytoplasmic RNA viruses such as dengue and chikungunya have epitranscriptomic modifications in their RNA. “Two groups showed in several papers how the m6A modification occurs in these viruses. These papers were highly cited, and in the years since, a lot of money has been invested in studying this and other cytoplasmic modifications in these viruses,” explains Díez. The unanswered question was how these modifications could occur when these cytoplasmic viruses never enter the nucleus, where the machinery to make them is located.

This is what both teams were wondering when they started to look at these modifications in more detail. “We started the project looking for the m6A modification, assuming, like everyone else, that it existed,” says the virologist. Using the chikungunya virus, which is expressed at very high levels, as a model, they repeated the technique that had been used in the original papers, based on an antibody that recognises the RNA modification they were looking for.

“The postdoc Belinda Baquero and the PhD student Mireia Puig repeated these experiments, but adding some controls that we considered essential; to our surprise, we didn’t see the modifications,” recalls Díez.

The next step was to use the Nanopore system that Novoa’s team had developed. In this case, they also used the necessary controls, with the synthetic transcript of all the viral RNA. Again, the result was negative.

They then decided to try a third, more sensitive method called Select. This allowed them to look specifically at a particular location in the RNA, but again they did not see the modification described.

Using three different methods, and with many positive and negative controls, the laboratories did not observe RNA modification.

The conclusion was clear: the m6A modification did not occur in this RNA virus. “The technique used initially, with the antibodies, gives a lot of background noise… And sometimes it does seem that you can see something if you lower the threshold enough, if you consider that they occur at very low levels, but the question is, ¿is it reproducible?,” Novoa asks.

Swimming against the tide

“Obviously, it is much more difficult to prove that something ‘doesn’t exist’ than to prove that it does. But we’ve done a lot of checks, we’ve looked at three different methods… we’re very confident that this m6A modification doesn’t exist,” explains Díez. “This study shows how important it is to use orthogonal methods, to look at things in many different ways, and to use extensive controls,” she adds. Novoa agrees: “A recent paper on RNA modifications – nothing to do with viruses – mentioned precisely that, especially in this field which is now being given a lot of importance, you have to be careful with false positives…. It’s very easy when you see something that you expect, to quickly assume it’s real. But you have to use good controls, in this case synthetic RNA, to prove that what you detect is true”.

“It’s very easy when you see something, to quickly assume it’s real, because it’s what you expect… it’s important to look at things in different ways and with good controls”.
Eva Novoa, CRG

When asked if it was difficult to publish a result that was so contradictory to what was hitherto believed, the two scientists replied that the reviewers of the paper quickly realised that it was a much-needed study. “There had already been doubts in the field about this finding for some time… But not enough to stop investing in it!”, says Díez.

However, the process has been hard. “At the beginning you doubt your own methods and techniques. You wonder what you are doing wrong, why the method is not working well… But in the end we have done a strong study, with many controls and complementary methods, which has allowed us to clarify the epitranscriptomic picture of cytoplasmic viruses,” concludes Novoa.