You'd Prefer An Argonaute

I didn’t go to this meeting, but it sounded like there were fewer marquee talks this year, due in part to the host of other small RNA meetings recently. Plus it was raining cats and dogs in Monterey. Thus the “Enhhh.”

Still a few nuggets worth mentioning though.

Ian MacRae told an interesting result of guide strands being unloaded more with increased complementarity, especially in the 3′ end, to the target RNA. Introducing more mismatches could inhibit unloading. Therefore when targets are present, miRNAs are more stable in AGO than siRNAs, possibly due to the acrobatics that AGO performs to accomodate a ~19-21mer duplex, especially the PAZ domain which interacts with the 3′ end of the guide. I imagine the degree of this effect is also AGO specific.

The structure of C3PO, an endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products, was solved twice and was compared to an avocado and the Death Star. Yay.                          

There was a bit of controversy regarding a paper published by Rachel Green’s group early last year which proposed an allosteric site in the MID domain that could bind the 5′ cap of mRNAs (which I presented in RNA Journal Club and reviewed here). Filipp Frank from Nahum Sonenberg’s group presented convincing biochemical and structural studies that showed no functional evidence for this allosteric site in the MID domain.

Argument over the mechanism of miRNA repression–mRNA degradation vs. translational repression in both temporal and absolute senses continued to be hotly debated.

Elisa Izaurralde pushed the idea that deadenylation is a direct effect of miRNA action, and not solely a consequence of translational repression.

One of the most intriguing talks came from Antonio Giraldez. His lab has been doing ribosome profiling in zebrafish during early embryogenesis where miR-430 dominates to clear maternal mRNAs, so the system is pretty clean. His main point was that in this scenario, at one time point where miR-430 expression peaks, there is translational repression without mRNA degradation. At the next time point a couple hours later, mRNA degradation comes into play, perhaps even slightly more than translational repression. Understanding the kinetics of deadenylation in relation to mRNA degradation will be key to interpreting these data from time points so close to miR-430 expression peaking.

Lastly, lush biochemistry is still the essence of the Yuki Tomari lab. They have some nice in vitro assays to follow piRNA processing, namely trimming of precursor molecules by a yet unidentified nuclease whose function seems to be coupled with methylation of the piRNAs.

The Non-Coding Genome meeting held at EMBL in Heidelberg this past October was great. To me, this “European Keystone” bettered the two (American) Keystones I’ve attended (’08 and ’09), with more breadth, including prokaryotic regulatory RNAs and lincRNAs. NCG also ran a more functional schedule, with the full day comfortably broken up with ample coffee/tea and meal breaks. The food was also ten-times better, and the venue was nifty in the building interior boasting a true double-helical walkway. Neat. This year’s organizers mentioned they plan for NCG to continue bi-annually, so look out for it in twenty-twelve.

After Heidelberg I hurried down to Stuttgart to get my car fix at the impressive museums of Porsche and Mercedes-Benz (highly recommended even if you have only a casual appreciation for German autos). Next, in a blur of French countryside, I was whisked to Paris, where everything was lovely. Everything. The art, architecture, churches, food, parks, the ladies. I spent my visit there with a friend, former labmate, and esteemed blog contributor (Robin!) who just started a Postdoc in Paris at the Pasteur. To those with true visual and gustatory aesthetics, Paris will make you weak in the knees.

Sometimes good things happen to Ph.D. students!

Heidelberg, Germany

You know those helixes that are, like, double helixes?

Porsche Museum, Stuttgart, Germany

View from Eiffel Tower

Crepe making in Paris

Paris skyline as seen from Centre Pompidou

Stopping over in Iceland

Heading home

Outside the IMBA (site of conference)

In a black+tinted Audi A8, briskly driven toward our plush hotel in central Vienna by a sharp looking Austrian, my trip began. These Europeans, they live properly. My 007-like arrival, however, soon gave way to an intense urge to sleep. But before I hit dreamland, with my friend I managed a trek up the tower of the ~14th century Gothic cathedral Stephansdom. Our reward later was a full meal, and cold lagers. Deep sleep was now imminent, and would do me well until next morning: the start of the 5th Microsymposium on small RNAs. It was a damn fine conference.

MicroScientists working at IMBA

After attending two consecutive Keystone RNAi conferences (2008-2009), it was so refreshing to be at a meeting where I didn’t feel so anonymous. Due to the smaller size, and perhaps partly the fact that half my lab wasn’t there (thank god!), there were numerous opportunities to meet other students, postdocs, and PIs. The conference hosts were friendly, and the meals were very good. The meeting ran smoothly. For entertainment, the second night there was a great Brazilian drums-guitar duo that provided pleasant rhythms. And good times were had by all out at the bars. They don’t close at 2am in Europe. You can stay as loooonnggg as you want.

Auditorium at IMBA

The big-shots Narry Kim, Phil Zamore, Dinshaw Patel, and Olivier Voinnet all gave very nice talks. These, along a talk by fast rising Yuki Tomari, were the highlights for me. But the talks by numerous junior group leaders are what really set the tone for the meeting. Young blood ruled here. Nine talks by Ph.D. students further cemented this feeling. (I was fortunate to give one of them.) By contrast, now I recall Keystone speakers as comparatively geriatric.

In terms of topics, animals, plants, yeast, and bacteria were all discussed. Along with a panoply of methods, from deep sequencing to whole animal, all the bases (pun intended!) were covered. Applause for Javier Martinez especially, as well as Julius Brennecke, for constructing such a great program. Overall, in terms of the science, food, schmoozing, and boozing–the 4 cornerstones of a meeting–Vienna hit a home run.

After the conference, my beer-laden hops (pun intended!) up to Prague and then Munich nicely rounded out my central European vacation. Below are some photos from my digital. I actually shoot primarily with a 35mm camera, and have many nice photographs from this trip on film, many of which will end up eventually here.

And now my awards for the cities of Vienna, Prague, and Munich:

• Best Science: Vienna (no contest!)
• Best Beer: Prague (for god sakes man, it was cheaper than bottled water, and soooo delicious.)
• Most Beautiful City: Prague
• Most Beautiful Women: Vienna
• Best Museums: Both in Munich, the splendid Brandhorst (modern art), and, vroom-vroom, the BMW museum!

Pictures from Prague, and the BMW Museum in Munich:

Prague

Týn Church and Old Town Square, Prague

St. Vitus Cathedral, Prague Castle

BMW R 32 motorcycle (1923-1926)

BMW 502 3,2 Liter Super (1957-1961) wheel, reflecting me and a 3.3 Li (1975-1977)

Sexy engine detail, 1997 BMW S50 B32.

Time to break out the shorts and Ray-Bans! I’m going to Europe. Well actually, I’m going to Vienna–which is looking extremely lovely–to attend the 5th Microsymposium on Small RNAs, to be held at the IMBA May 17-19. (Don’t worry, I’m not bringing the shorts, and have never owned Ray-Bans or an American-abroad-arrogance.)

This place and this conference are right up my alley. It’s gonna be hella sophisticated. Scarfing down topfenkolatsche and Ottakringer while chatting up the impressive speakers/attendees, that is the goal. How do scientists schmooze in Europe? Probably with better food and more cigarettes. And ohhh, the bier... you’re the other reason I’m coming.

Never having been to Europe before, my lab mate remarked, “you’re going to lose your European virginity!” Well damn, it’s about time.

Stories and photos forthcoming.

Look to the plant. Look at ’em; eat ’em; use ’em for medication; and appreciate them because they offer numerous biological fruits, including well studied RNAi. Here are some things overheard at this year’s RNA Silencing Mechanisms in Plants Keystone Symposium, which met in Santa Fe late last month. (Thanks a bunch to my friend M. for helping with this.)

A couple heavy-hitters described a class of predominantly 22 nt miRNAs in Arabidopsis, Dcr-like 1 products, that initiate secondary siRNA biogenesis by an unknown mechanism. This has also been seen in rice, where they are also Dcr-like 4 dependent. They key point is that specific miRNAs only appear to be able to initiate secondary siRNA biogenesis if they are 22 nt, and not 21 nt. (Most miRNAs in Arabidopsis are 21 nt.) The implication is that Argonaute can decipher a 1 nt difference in length of a miRNA, and this may induce some structural change that could, for example, lead to recruitment of RdRP and other factors responsible for producing secondary siRNAs. Pretty fascinating that such a minute difference could be responsible for such critical decision making.

Another speaker presented work showing some elegant, and very technically challenging, grafting experiments in Arabidopsis that show 24 nt siRNAs can move through tissues, and cause RNA induced DNA methylation. [Hearing this caused me flashbacks to my first-year grad Development course in which we learned about pioneering grafting experiments in newts in the early part of last century, and how crazy it is that these Franksteinian techniques work so well.] Using a different technique, particle bombardment, another speaker demonstrated spreading of siRNAs through cells. Very fascinating to think of how spreading is regulated/controlled to remain specific. They’d be potent little things to be sending out without exact destinations/address labels.

Although it is worth pointing out that 24 nt siRNAs repress transposons and repeat elements, so it may not be so bad to have this activity be somewhat constituitive in a given region when such a challenge is detected. When silencing components were removed, transposition was observed to grow slowly. When silencing components were re-introduced, it took several generations to re-establish silencing, in reproductive tissues.

One of the above speakers also described experiments mating tomatoes. [I love tomatoes myself, and, did you see The Simpsons episode where Homer see’s Ralph Wiggum’s alcohol powered car at the Springfield Elementary science fair? “One for you; one for me” Homer says, as he dreams sipping half of his fuel purchase. That would be me if I was mating tomatoes in lab.] What was really cool is that after a few generations they saw the emergence of new siRNAs that were neither present in the parental nor F1. These progeny with novel siRNAs expressed new traits as well. Maybe I’m overinterpreting the result, but, wow! It’s unlikely perhaps, but the traits could be solely due to the fresh siRNAs. Or more interestingly, could evolution be dictating some sort of package deal for the emergence of new traits? “I’ll give you a new trait, but you’ve gotta take small RNA(s) along with it.” Or maybe it’s simply coincidence–you mate two non-isogenic things, and all kinds of stuff comes out.

Also mentioned was a new set of 24 nt miRNAs in rice, Dcr-like 3 dependent, present in reproductive organs, that direct methylation of their target genes in trans, in a strand specific manner. Yeah, but what kind of rice? Was it brown basmati?

Finally, comparing two different Arabidopsis strains, one Columbia (from Missouri) and one from the Cape Verde islands, a speaker described that they produced vastly different genome-wide Cytosine methylation patterns. Promoter methylation appeared to be conserved while gene body methylation was greatly reduced in the Cape Verde Island strain. Presumably this has some significant functional consequence, perhaps due to the differential environmental responses that the two strains have evolved.

I wish I was in Cape Verde right now, on the beach, eating a salad.

I didn’t make it to this year’s Keystone conference, RNA Silencing: Mechanism, Biology and Application, held last month in Colorado. I know, however, a bunch of people that did go, and they said it was pretty good. Pre-tay, pre-tay, pre-tay, pretay good.

Some things I heard (thanks kindly to my comrades):

A theme that first splashed at last year’s Keystone was a major interest this year: CLIP experiments. One surprise observed in these datasets is much more Argonaute binding in open reading frames than was previously considered. Some extend this to say that there is much more functional binding in ORFs than was previously thought. I imagine, however, that much of this binding is non-specific, or transient, analogous to what’s seen for transcription factor binding genome wide (e.g. see Li et al. 2008). Prudent calibration of potential background/noise should be required before the outstanding potential of these immense datasets can be exploited to make fresh, solid conclusions about miRNA targeting. One group is extending their CLIP method beyond just Argonautes, and looking for the RNA binding signatures of many other RNA binding proteins.

One speaker revealed a triumphant result that nicely explains why knockouts of mammalian Ago2, but not the other Argonautes, are embryonic lethal. This was previously very confounding because while mammalian Ago2 is the only Ago with slicer activity, slicing activity hasn’t been widely demonstrated in mammals. All four mammalian Agos obviously facilitate miRNA seed directed repression, but now we have a thoroughly satisfying explanation for why slicer activity is also necessary in the developing embryo.

A couple groups showed some nice new structural and biochemical data that further refines our understanding of which domains of GW182/TNRC6 and PABP bind each other. Early looks show some predicted binding differences in two model systems studied, fly and mammal. The data is revealing more about the mechanism of repression of mRNAs by miRNAs.

Lastly, another speaker convincingly showed why some sRNAs are methylated and some are not. In plants, for example, the vast majority of target sites form near perfect complementarity with miRNAs, and all the miRNAs are methylated. The same goes for endo-siRNAs in flies. But in mammals, the vast majority of miRNAs do not have perfect site targets, nor are they methylated. Methylation appears to provide protection from degradation of the sRNA when it pairs with a perfect site target. In contexts where many perfect sites for sRNAs are present, sRNAs will be methylated. In contexts where such sites are very rare, the sRNAs aren’t methylated. A very pleasant resolution.