Arms race gains a third layer of counteradaptive complexity

The RNAi pathway (covered before here) is a major immune mechanism in plants and invertebrates, but it can be suppressed by the very viruses it is trying to stop.  The virus produces an RNA Silencing Suppressor (RSS), a protein that binds or degrades proteins in the RNAi pathway, giving the virus free reign to replicate as it pleases.  This has the potential to create a fast-moving arms race: host suppresses virus using RNAi, so virus suppresses host suppression mechanism, so host suppresses the viral suppressor of host suppression…

Only the first two steps in this cycle have been found so far.  The RNAi machinery has been extensively documented, and many viral RSSs have been catalogued.  However, a recent paper in PNAS by Nakahara et al may have found the first instance of the third step in the cycle: a host suppressor of RSSs.

The authors focused on rgs-Cam, a tobacco plant protein that has previously been observed to interact with viral proteins (Anandalakshmi et al, 2000).  They found that it binds RSSs with greater affinity if they have an arginine-rich domain.  This domain is also what the RSS uses to bind small interfering RNAs (a constituent of the RNAi pathway), meaning that rgs-Cam may be specifically targeting only those proteins that suppress RNAi.

To test its effect on the activity of RSSs, the authors depleted rgs-Cam in tobacco (ironically by using RNAi to knock it down).  This led to increased suppression of RNAi by two RSSs (2b and HC-Pro).  They then created transgenic plants with either increased or decreased levels of rgs-Cam.   They found that those with more rgs-Cam had reduced RSS activity and were less susceptible to viral attack, whereas those with less rgs-Cam had increased RSS activity and were more susceptible to attack by viruses.

So it looked like rgs-Cam suppressed RSSs, and therefore restores the RNAi response.  But how?  The authors inhibited different cellular pathways and found that when the autophagy pathway was inhibited, the levels of rgs-Cam and RSSs both increased.  From this they concluded that once rgs-Cam binds an RSS, both are broken down by autophagy-like protein degradation (ALPD).

Interestingly, the authors highlight previous work that reported an inhibition of the RNAi mechanism by high levels of rgs-Cam.  This causes them to speculate that rgs-Cam may be triggered in emergencies only.  When the normal RNAi pathway can deal with the virus, rgs-Cam levels are kept low; however, when the virus gets out of hand, rgs-Cam is upregulated (or de-inhibited, the authors are admirably honest about the remaining ambiguity in the exact mechanism).

Role on the discovery of the first VSHSRSS – Viral Suppressor of Host Suppression of RNA Silencing Suppressor!

References

Nakahara, K.S. et al (2012) Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppresors.  PNAS Early Edition: 1-6.

Anandalakshmi, R. et al (2000) A calmodulin-related protein that suppresses post-transcriptional gene silencing in plants.  Science 290: 142-144.

Evidence mounts for antiviral piRNAs

piRNAs are a type of small RNA in the RNAi mechanism.  Current thinking limits them to an anti-transposable element role in the germline; however, a new paper in PLoS One by Vodovar et al adds to the growing body of evidence that this strict classification may be an oversimplification, and that piRNAs may have an antiviral role.

There are two things that distinguish piRNAs from other small RNAs: they are 25-30 nucleotides long, compared to siRNAs which are 21nt long; and they have a bias for uridine at their first position and adenosine at their tenth.

So to find if piRNAs function against viruses, the authors injected cells from the mosquitos Aedes albopictus and Aedes aegypti with Sindbis Virus (SINV), and extracted and sequenced the small RNAs produced in response.

They found small RNAs of 21 and 25-29 nucleotides in length (displayed below): these are the appropriate sizes for siRNAs and piRNAs respectively. (all figures adapted from Vodovar et al, 2012)

Then they looked at whether the sequences of these small RNAs corresponded to anywhere on the SINV genome, and found matches across the genome: it is therefore likely that these small RNAs were derived from the viral genome.  This is displayed below: the higher the bar, the more small RNAs mapped to that location.

Thirdly, they sequenced the small RNAs and looked at how biased each position was (i.e. whether A, C, G or U occurred more than 25% of the time).  They found a strong bias for U at their first position, and A at the tenth position, characteristic of piRNAs (displayed below, with the size of each letter corresponding to its frequency relative to the other three bases).

They confirmed this finding by analysing the data from an earlier study by Brackney et al on A. albopictus infected with La Crosse Virus (LACV), and found the same U and A biases in small RNAs in the same 25-29 nucleotide size range

Finally, they checked whether piRNAs in these mosquitos are still fulfilling their original function of defence against transposable elements.  They did this by extracting small RNAs extracted from A. aegypti cells under normal (i.e. no virus present) conditions, and mapping these to the A. aegypti genome sequence, which contains sequences for the transposable elements in this species.  They found the same results as during viral exposure: peaks at 21 and 25-29 nucleotides long; a first position U and tenth position A bias; and matches to the sequences of many different transposons.

This paper presents convincing results for piRNAs being both an antiviral and anti-transposable element mechanism in mosquitos.  While they only present data from cell culture, a previous paper by Morazzani et al (2012) has found a similar phenomenon in whole mosquitos, thus increasing the confidence with which these conclusions can be extended to natural insect immune responses.  Importantly, the authors find the same results using cells with a normal RNAi mechanism and cells deficient in Dcr-2, a protein integral to the siRNA pathway: it seems that this piRNA production is not merely a back-up for the siRNA response, but a response in its own right.  Their discussion also highlights vertical (mother to offspring) viral transmission in arthropods: could the presence of vertically transmitted viruses in the germline have been the selective pressure that drove an antiviral function in the piRNA pathway? And does this limit the prospect of finding antiviral piRNAs in other animals, which don’t transmit viruses vertically?

 

 

References

Vodovar, N., Bronkhorst, A.W., van Cleef, K.W.R, Miesen, P., Blanc, H., van Rij, R.P. and Saleh, M-C (2012) Arbovirus-derived piRNAs exhibit a Ping-Pong signature in mosquito cells.  PLoS One 7: 1-8

Morazzani, E.M., Wiley, M.R., Murreddu, M.G., Adelman, Z.N. and Myles, K.M. (2012) Production of virus-derived Ping-Pong-dependent piRNA-like small RNAs in the mosquito soma.  PLoS Pathogens 8: 1-11

 

Introducing…RNAi

Nucleic acids (DNA and RNA) underpin everything in biology.  All the information about an organism is stored in nucleic acid form; and all the actions that are taken from this information rely (directly or indirectly) on nucleic acids.  So it should be no surprise that a system has evolved that allows one individual directly to manipulate the nucleic acids of another.  But the dual simplicity and intricacy of this system are truly astounding, all the more so as new discoveries are rapidly made.

The system is RNA interference (RNAi).  It is based on two simple concepts: extreme specificity in recognising its RNA targets, and generality in the mechanisms that deal with these targets.  Though it was only discovered (somewhat unwittingly) by Napoli et al in 1990, it has since been comprehensively characterised (resulting in the 2006 Nobel Prize being awarded to Andrew Mello and Craig Fire).  While its mechanisms and functions vary between different groups and even kingdoms, the basic idea is as follows:

 

The generality of the system comes from the large proteins that do the cutting: just a few different proteins can be recruited to degrade a wide array of targets.  And the specificity is conferred by the small RNAs: because they are derived from the target itself, they will necessarily be specific to that target.

It is the fourth step in the mechanism – where RNA derived from the target guides the rest of the machinery to the correct place on the correct target messenger RNA (mRNA) – that makes the system so effective and economical.  Regardless of mutations that occur in the target’s genome, the cell will always be able to deal with it, as it will use this newly mutated sequence to guide its machinery – the target can never escape degradation simply by mutational change.

These small RNAs can be broadly classified into three types.  Small interfering RNA (siRNA) targets extracellular parasites such as viruses, and hence is integral to the immune system.  Piwi-interacting RNA (piRNA) targets intracellular parasites like transposable elements (sections of the host’s own genome which can cut themselves out, or copy their sequence, and paste this back into the host’s genome), and is thought to be expressed exclusively in the germline (reproductive cells) where these elements are most active.  And microRNA (miRNA) targets the host’s own gene transcripts, allowing it to fine-tune gene expression.

These different small RNA types mean that the RNAi pathway has a dizzying array of functions in different organisms: stem cell maintenance, chromatin formation and upkeep of fertility to name just a few, as well as the antiparasite role mentioned earlier.  They have also become a standard tool for investigations into gene function and expression: by injecting the right double-stranded RNA sequence into a cell, one can knockout a specific gene while keeping everything else constant.  By seeing what the cell can no longer do, or what it starts to do, reliable inferences can then be made as to this gene’s function.

However, recent work is calling into question the strict delineation of function between the different small RNA types.  Interactions between pathways and crossover in their functions are blurring the boundaries between siRNA, miRNA and piRNA.  Most excitingly, new work is building the case for RNAi occurring in humans: future discoveries, and applications of these discoveries, will be truly fascinating.

References

Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans.  The Plant Cell 2: 279-289

Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.  Nature 391: 806-811

Okamura, K. (2011) Diversity of animal small RNA pathways and their biological utility.  WIREs doi: 10.1002/wrna.113