Since the discovery of the first microRNA a decade ago, more than two hundred of them have been identified in humans. Although a lot of the work on miRNAs involves trying to uncover their roles in the cell, Duke University researcher Bryan Cullen has taken an interest in answering the more fundamental questions of how miRNAs are made and how they work.
Cullen told RNAi News that he has not dedicated much time to the determination of miRNA functions because it “really is an exercise in making knockout mice and seeing if they look different from regular mice, which isn’t really our thing.” As such, he has focused his attention on what he describes as the basic end of the business.
“The thrust of the [research] deals with biogenesis of microRNAs, specifically: How are they transcribed? What transcribes them? What are the features of the precursors that make them susceptible to accurate processing by Drosha? What regulates the ability of those RNAs to interact with the Exportin-5 nuclear export factor? How [does this] play out in the cytoplasm in terms of Dicer processing and loading into RISC,” he said.
Cullen isn’t alone in pursuing answers to these questions, “but my emphasis has very much been on the whole issue of human microRNAs,” he said. Researchers such as Phil Zamore of the University of Massachusetts Medical School and Dave Bartel from the Whitehead Institute, who have made significant discoveries regarding the mechanisms by which miRNAs work, “all are working in Drosophila and C. elegans,” Cullen noted. “But our sense is that, in the end, we really want to know how the system works in human cells.”
According to Cullen, it is becoming clear that RNAi operates in significantly different ways in different organisms. “I think that’s still causing quite a bit of confusion. There’s not a lot of evidence, as far as I’m concerned, in human cells that RNA interference plays out at the level of transcriptional regulation.” Additionally, unlike in Drosophila where there are two Dicer enzymes, which mediate miRNA and siRNA cleavage, respectively, “in humans there’s only one enzyme that does both,” he said. “We and others have published data suggesting that in the human system there really is no difference between microRNAs and siRNAs.”
Cullen said that he has concluded that the miRNA pathway “is the system in the human cell. In my view, that’s the only pathway that exists.” In the June issue of Virus Research, Cullen wrote as much, noting that the post-transcriptional regulatory machinery used by siRNAs and miRNAs is largely or entirely identical in vertebrate cells.
“MicroRNAs and siRNAs are different only in where they come from — microRNAs being encoded within the genome and siRNAs being, generally speaking, exogenous,” he said. “This makes it very important to understand how the microRNA pathway works.”
In a paper published in Genes and Development in December, Cullen and his colleagues at Duke demonstrated that Exportin-5 is required for the exportation of pre-miRNAs — the roughly 65-nucleotide long RNA hairpin intermediates processed in the nucleus from long transcripts — to the cytoplasm where they are processed into miRNAs.
His current work — being funded by a four-year National Institute of General Medical Sciences grant, worth about $760,000 — seeks in part to build off this discovery by defining the characteristics of pre-miRNAs that mediate their binding to Exportin-5.
Cullen also aims to define the RNA sequences or structures that mediate the specific nuclear excision of the pre-miRNA from [an] initial transcript; define the features of pre-miRNA that mediate the specific cytoplasmic excision of the mature miRNA by Dicer; understand how miRNAs cooperate to inhibit mRNA translation; and understand how endogenous miRNA stability and function is affected by the introduction of exogenous siRNAs, according to the grant’s abstract. Additionally, Cullen hopes to extend initial data on the inhibition of miRNA function by adenovirus VA1 non-coding RNA by trying to define this phenomenon’s underlying mechanism.
“We’ve also been working a lot on what Drosha recognizes,” he added. Cullen declined to comment in detail about this aspect of his work given its preliminary nature, but said that “it recognizes particular structures rather than sequences. That’s perhaps why all the microRNAs, even though they look so completely different to one another in primary sequence, can still all be recognized by the same enzyme.”
Cullen’s latest miRNA project follows some proof-of-principle experiments using siRNAs to silence HIV. While these efforts were successful in the sense that lentivirus vectors expressing siRNAs specific for the transcription factor Tat or the cellular co-receptor CCR5 were able to block HIV-1 replication in vitro, Cullen has since concluded that RNAi would not make a viable therapeutic against the virus.
“My perception is that the virus becomes resistant to siRNAs so quickly that it’s a futile endeavor,” he said. “If you just crunch the numbers, it’s pretty pointless.” According to Cullen, there are about one billion HIV-infected cells per individual, and the size of the HIV-1 genome is about 10,000 nucleotides.
“We also know that the reverse transcriptase of HIV makes one mistake per genome replication cycle, so it makes an average of one mistake for every 10,000 nucleotides it copies,” he said. “That means … every single mutation that could possibly exist in HIV-1 at the single nucleotide level is being generated 100,000 times a day.”
As a result, Cullen contends that right off the bat there are between 100,000 and 1 million viruses in any given infected person that are already resistant to a particular siRNA.
He doubts, too, that multiple siRNA-based drugs would work. According to Cullen, HIV-1 becomes resistant to existing treatments by changing multiple amino acids in the enzymes the drugs are targeting, a move that is significantly more complex than “simply changing individual nucleotides.” As such, Cullen said, “even by hitting simultaneous targets that are conserved, my own perception is that you’re still not going to get anything accomplished.”
On top of this, HIV-1 is a highly disseminated disease, Cullen noted, and T-cells “are not particularly prone to take up nucleotides.”
Cullen said he does, however, remain excited about using RNAi as a research tool for HIV. “We used a cloned HIV-1, so that we wouldn’t have to deal with all the sequence variation, and I was impressed with it … as a tool to identify, for example, genes that are very important in the HIV lifecycle.”
Cullen said that by conducting siRNA screens, it could be possible to identify traits that make cells more resistant or receptive to HIV-1, or find genes that HIV-1 uses or interacts with, all of which could result in potential druggable targets. “From my perspective, that’s where the potential of RNAi lies in the HIV-1 field — as a tool,” Cullen said.
“HIV-1 is probably one of the least promising diseases that one might think about” for RNAi-based therapy,” he said. “But I’ve been wrong before.”