Sunday, June 15, 2008

Capped Small RNAs Expand Universe of Small RNAs

Findings from research by myself and colleagues here in Stanford on the discovery of a new species of distinct small RNAs, capped small RNAs, have just been published online at Nature Structural & Molecular Biology (Haussecker et al.: Capped small RNAs and MOV10 in human hepatitis delta virus replication).

It is becoming clear from large-scale sequencing efforts and hypothesis-driven research such as ours, that siRNAs and microRNAs are just two classes in an ecosystem of other small and non-coding RNAs. This particular class of small RNAs carries mRNA-like 5’ cap structures and was discovered during our studies on human Hepatitis Delta Virus (HDV) replication.

HDV is the smallest virus known to the animal kingdom and is even more so remarkable in that it does not encode for its own polymerase for viral replication, as all other viruses do, and instead relies on host RNA Polymerase II (Pol II) for its replication. HDV accomplishes this with carrying the genetic information for just one non-catalytic protein, the hepatitis delta antigen (HDAg), while its RNA genome calls all the other shots. It is also the only known example of RNA-directed transcription by Pol II in vertebrates, as Pol II is largely thought to use DNA, not RNA, as a transcription template.

Intrigued by this and by the possibility that within the complexity of our transcriptome there may be hidden RNA-directed transcription, vestiges of our RNA World heritage, and HDV may be the key to its understanding, we set out to investigate whether small RNAs would also play a role in HDV replication. Sure enough, a few Northern blots later, it became clear that RNA secondary structures previously associated with the initiation of HDV transcription harbored small RNAs. Long story short, the occurrence of capped small RNAs from hairpin secondary structures suggests that maybe analogous RNAs in our genome could be involved in analogous processes.

As we were studying the capped small RNAs, we also sought to determine the host factors that HDAg interacted with. Knowing this may give us further insights into HDV-related RNA-directed transcription. Intriguingly, the mass-spectrometry screen yielded MOV10 which is a gene that in plants had been implicated in RNA amplification during RNAi. Knocking down MOV10 with siRNAs inhibited HDV RNA accumulation, consistent with a function in RNA-directed transcription in vertebrate cells as well. Given that triphosphorylated small RNAs, which like capped small RNAs, should be derived from short transcription initiation events, had been found during RNAi amplification in worms, we speculate that HDV replication may indeed tap into an evolutionarily conserved process which makes it also more likely that non-viral RNA-directed transcription indeed occurs in our cells.

While RNAi-related factors such as Dicer and Drosha do not appear to play a role in HDV replication, we found in the course of our studies that knockdown of Argonaute 4 (AGO4) significantly affected HDV replication. AGO4 is related to AGO2 which is the Slicer in RNAi that cleaves target mRNA, and yet very little is known about AGO4. Determining the roles and preferences of the various AGO proteins in humans will be an important area of RNAi Therapeutics research as it promises to yield improvements in the design of RNAi triggers both in terms of safety and knockdown potency.

It remains to be seen whether capped small RNAs are specific for RNA-directed transcription, or whether they are a reflection of a more widely used gene regulatory mechanism. In fact, high-throughput sequencing efforts by others indicate a population of small RNAs associated with gene promoter regions. It is possible, although not yet demonstrated, that these are capped, and since the HDV small RNAs are abundant enough to be detected by Northern blot, HDV replication may serve as a model system to understand this type of gene regulation. Finally, it is tempting to speculate whether the modulation of capped small RNAs may be utilized for therapeutic purposes.

4 comments:

Anonymous said...

Dirk

congratulations on your publication and discovery.

It makes one wonder how many more cellular processes involving RNA with potential future therapeutical applications remain yet to be discovered.

It also demonstrates how research of cellular mechanisms in lower evolutionary forms may directly benefit therapeutical research in that the same or similar mechanisms may be conserved in human cells but concealed and expressed only in specific host/pathogen interactions.

And thanks for your blog summary as I confess I wouldn't have the faintest idea what your paper is about by simply reading it (or attempting to do so)... and they complain that legal writing is incomprehensible ;o)

Further to the discussion a couple of posts below, do you know if ALNY ever released any information on how the ALN-RSV01 molecule passes thru the cell membrane and into the cytoplasm? They claim to have ruled out non-specific response by testing against control siRNAs, but in light of the scathing Nature article on the non-specific mechanism of action of the naked siRNA used in the AMD model, I keep wondering what is it that makes the lung cells different in terms of naked siRNA uptake.

Martin

Anonymous said...

Congratulations -- that is a fascinating and exciting discovery. It shows that by revisiting the simplest of model systems, an "organism" that encodes one gene, one can make insightful discoveries.
-Derek

Dirk Haussecker said...

Thanks. It's surprising that the "lay" public here understands the value of simple model systems for biological investigation that, on the surface, do not directly impact on human health, while the "experts" deciding on grants often underappreciate this. Ironically, Hepatitis Delta Virus research has really suffered from the fact that it's medical importance has become less in recent years, particularly in the West. So I hope that this paper will be able to stimulate some additional interest in HDV research, as I believe it to be such a unique opportunity to study RNA-directed transcription.

Martin- I agree with you that there are better ways to deliver siRNAs to the lung than through the inhalation of naked siRNAs (such was demonstrated in the lipidoid paper, e.g.). However, it is clear from the literature, from many groups, that epithelial mucosa, such as in the lung, have the ability to take up some unformulated siRNAs, although it is not clear how. I also agree, that it is well possible that there is some immune contribution to the antiviral effect, although Alnylam did not find evidence for this in the experimental infection study. I will maybe write in another blog entry how one might turn the immunostimulatory lemon into lemonade by harnessing the potential dual biological activities (gene silencing and immunestimulation) of certain, not all, siRNAs. Alnylam (vaccine spin-out) and Tekmira have indicated interest in that area, which may turn out to be a quite significant opportunity, and I believe Pfizer/Coley and others may also have similar ideas.

Whatever the mechanism, at this point, as long as ALN-RSV01 is safe and efficacious it's fine by me. Indeed, it turns out that most blockbuster drugs have unanticipated secondary effects that turn out to be quite essential for efficacy. For RNAi Therapeutics, it's important to learn what to look for and manage such class effects, e.g. by dialing up or down the immune response as needed.

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