Protocells YAY!

I’m briefly surfacing from the stress ocean that is paper writing to do a little dance of joy about the latest mind-blowing development in origin-of-life research.

(With my ability to go on endlessly about random scientific subjects, you’d think I’d love writing papers. No, no, no, hell no. I wish I could just upload my methods and figures to some database and be done with it. >.<)

My latest great squeal about abiogenesis research was due to an RNA enzyme that could copy long RNA strands. Well, that’s still bloody amazing, but maybe massive RNA enzymes are not how the thing we call life started. Jack Szostak’s group works witn a model of early life in which enzymes aren’t needed at all.

They’ve been working for years and years on their protocells (illustration above by Janet Iwasa via exploringorigins.org). These are basically little fat bubbles floating around in a watery solution, with a bit of nucleic acid inside. The fatty membranes of protocells are made of much simpler materials than modern cell membranes. Protocells haven’t got any proteins, and contain just a tiny “genome” that doesn’t encode anything meaningful. Yet they can, under the right circumstances, grow and divide and pass on that genome to their descendants through ordinary physical forces.

And now, they can also copy it.

The problem so far was magnesium. RNA can be replicated by an enzyme, or it can, to an extent, copy itself using base pairing. Magnesium is necessary for both kinds of replication. However, the Szostak group’s fatty protocells quickly fall apart in the presence of magnesium, spilling all their RNA content.

Adamala and Szostak (2013) tested a bunch of small molecules that bind magnesium to see if they could help. Many of them could protect the protocells, but only one, citrate, could do this without also stopping RNA replication. As a bonus, citrate prevented the degradation of RNA that, under normal circumstances, eventually happens at high magnesium levels.

Like other research toward RNA replication, this study isn’t quite there yet. For one thing, the “genomes” of these protocells are very limited – they are tiny, and they are just runs of a single RNA building block, so it’s hard to imagine how they could be precursors to more “meaningful” genomes. Also, although a lot of organic molecules just spontaneously show up when someone tries to recreate early Earth chemistry, citrate is not one of them.

Nonetheless, little by little we’re edging closer to a living RNA world. We may never know how life actually started, but the research on how it could have started looks more exciting by the day…

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Reference:

Adamala K & Szostak JW (2013) Nonenzymatic template-directed RNA synthesis inside model protocells. Science 342:1098-1100

A bunch of cool things

From the weeks during which I failed to check my RSS reader…

1. The coolest ribozyme ever. (In more than one sense.)

I’ve made no secret of my fandom of the RNA world hypothesis, according to which early life forms used RNA both as genetic material and as enzymes, before DNA took over the former role and proteins (mostly) took over the latter. RNA is truly an amazing molecule, capable of doing all kinds of stuff that we traditionally imagined as the job of proteins. However, coaxing it into carrying out the most important function of a primordial RNA genome – copying itself – has proven pretty difficult.

To my knowledge, the previous record holder in the field of RNA copying ribozymes (Wochner et al., 2011) ran out of steam after making RNA strands only half of its own length. (Which is still really impressive compared to its predecessors!) In a recent study, the same team turned to an alternative RNA world hypothesis for inspiration. According to the “icy RNA world” scenario, pockets of cold liquid in ice could have helped stabilise the otherwise pretty easily degraded RNA as well as concentrate and isolate it in a weird inorganic precursor to cells.

Using experimental evolution in an icy setting, they found a variation related to the aforementioned ribozyme that was much quicker and generally much better at copying RNA than its ancestors. Engineering a few previously known performance-enhancing mutations into this molecule finally gave a ribozyme that could copy an RNA molecule longer than itself! It still wouldn’t be able to self-replicate, since this particular guy can only copy sequences with certain properties it doesn’t have itself, but we’ve got the necessary endurance now. Only two words can properly describe how amazing that is. Holy. Shit. :-O

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Attwater J et al. (2013) In-ice evolution of RNA polymerase ribozyme activity. Nature Chemistry, published online 20/10/2013, doi: 10.1038/nchem.1781

Wochner A et al. (2011) Ribozyme-catalyzed transcription of an active ribozyme. Science 332:209-212

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2. Cambrian explosion: evolution on steroids.

This one’s for those people who say there is nothing special about evolution during the Cambrian – and also for those who say it was too special. (Creationists, I’m looking at you.) It is also very much for me, because Cambrian! (How did I not spot this paper before? Theoretically, it came out before I stopped checking RSS…)

Lee et al. (2013) used phylogenetic trees of living arthropods to estimate how fast they evolved at different points in their history. They looked at both morphology and genomes, because the two can behave very differently. It’s basically a molecular clock study, and I’m still not sure I trust molecular clocks, but let’s just see what it says and leave lengthy ruminations about its validity to my dark and lonely hours 🙂

They used living arthropods because, obviously, you can’t look at genome evolution in fossils, but the timing of branching events in the tree was calibrated with fossils. With several different methods, they inferred evolutionary trees telling them how much change probably happened during different periods in arthropod history. They tweaked things like the estimated time of origin of arthropods, or details of the phylogeny, but always got similar results.

On average, arthropod genomes, development and anatomy evolved several times faster during the Cambrian than at any later point in time. Including the aftermath of the biggest mass extinctions. Mind you, not faster than modern animals can evolve under strong selection – they just kept up those rates for longer, and everyone did it.

(I’m jumping up and down a little, and at the same time I feel like there must be something wrong with this study, the damned thing is too good to be true. And I’d still prefer to see evolutionary rates measured on actual fossils, but there’s no way on earth the fossil record of any animal group is going to be good enough for that sort of thing. Conflicted much?)

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Lee MSY et al. (2013) Rates of phenotypic and genomic evolution during the Cambrian explosion. Current Biology 23:1889-1895

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3. Chitons to sausages

Aplacophorans are probably not what you think of when someone mentions molluscs. They are worm-like and shell-less, although they do have tiny mineralised scales or spines. Although they look like one might imagine an ancestral mollusc before the invention of shells, transitional fossils and molecular phylogenies have linked them to chitons, which have a more conventional “sluggy” body plan with a wide foot suitable for crawling and an armoured back with seven shell plates.

Scherholz et al. (2013) compared the musculature of a living aplacophoran to that of a chiton and found it to support the idea that aplacophorans are simplified from a chiton-like ancestor rather than simple from the start. As adults, aplacophorans and chitons are very different – chitons have a much more complex set of muscles that includes muscles associated with their shell plates. However, the missing muscles appear to be present in baby aplacophorans, who only lose them when they metamorphose. (As a caveat, this study only focused on one group of aplacophorans, and it’s not entirely certain whether the two main groups of these creatures should even be together.)

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Scherholz M et al. (2013) Aplacophoran molluscs evolved from ancestors with polyplacophoran-like features. Current Biology in press, available online 17/10/2013, doi: 10.1016/j.cub.2013.08.056

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4. Does adaptation constrain mammalian spines?

Mammals are pretty rigid when it comes to the differentiation of the vertebral column. We nearly all have seven neck vertebrae, for example. This kind of conservatism is surprising when you look at other vertebrates – which include not only fairly moderate groups like birds with their variable necks, but also extremists like snakes with their lack of legs and practically body-long ribcages. Mammalian necks are evolutionarily constrained, and have been that way for a long time.

Emily Buchholz proposes an interesting explanation with links to previous hypotheses. Mammals not only differ from other vertebrates in the less variable numbers of vertebrae in various body regions; these regions are also more differentiated. For example, mammals are the only vertebrates that lack ribs in the lower back. In Buchholz’s view, this kind of increased differentiation contributes to adaptation but costs flexibility.

Her favourite example is the muscular diaphragm unique to mammals. This helps mammals breathe while they move, and also makes breathing more powerful, which is nice for active, warm-blooded creatures that use a lot of oxygen. However, it also puts constraints on further changes. Importantly, Buccholz argues that these constraints don’t all have to work in the same way.

For example, the constraint on the neck may arise because muscle cells in the diaphragm come from the same place as muscle cells associated with specific neck vertebrae. Moving the forelimbs relative to the spine, i.e. changing the number of neck vertebrae, would mess up their migration to the right place, and we’d end up with equally messed up diaphragms.

A second possible constraint has less to do with developmental mishaps and more to do with plain old functionality. If you moved the pelvis forward, you may not screw with the development of other bits, but you’d squeeze the space behind the diaphragm, which you kind of need for your guts, especially when you’re breathing in using your lovely diaphragm.

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Buccholz E (2013) Crossing the frontier: a hypothesis for the origins of meristic constraint in mammalian axial patterning. Zoology in press, available online 28/10/2013, doi: 10.1016/j.zool.2013.09.001

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And… I think that approximately covers today’s squee moments 🙂

A difficult landscape for the RNA world?

I’m back, and right now I can’t really decide if I should be squeeful or sad about Jiménez et al. (2013).

On the side of squeeing, I have some pretty compelling arguments.

1. It’s an RNA world paper. I’m an unabashedly biased fan of the RNA world. (Not that my opinion matters, seeing as that’s the only origin-of-life hypothesis I actually know anything about. It’s like voting for the only party whose campaign ads you’ve seen.)
2. I find the actual experiment ridiculously cool. It’s a bit like that mutation study about heat shock protein 90 that I wrote about aaaaages ago, except these guys evaluated the relative fitness of pretty much every single possible RNA molecule of 24 nucleotides. Yes, that is 4^24 different RNA molecules, each in many copies. And they did it twice, just to make sure they weren’t mistaking statistical flukes for results [1].
3. It explores the landscape of evolution and digs into Big Questions like, how inevitable/reproducible is evolution? Or, as Stephen Jay Gould would put it, what would happen if we replayed the tape of life?

On the other hand, the findings are a bit… bleak. So the experimental setup was to select from this huge pool of RNA sequences for ones that could bind GTP, which is basically a building block of RNA with an energy package attached. In each round of selection, RNAs that could attach the most strongly to GTP did best. (The relative abundances of different sequences were measured with next-generation sequencing.) The main question was the shape of the fitness landscape of these RNAs: how common are functional GTP-binding sequences, how similar do they have to be to perform this function, how easily one functional sequence might mutate into another, that sort of thing.

And, well.

1. There were only 15 fitness peaks that consistently showed up in both experiments. (A fitness peak consists of a group of similar sequences that are better at the selected function than the “masses”.) That sounds like GTP-binding RNAs of this size are pretty rare.
2. The peaks were generally isolated by deep valleys – that is, if you were an RNA molecule sitting on one peak and you wanted to cross to another, you’d have to endure lots of deleterious mutations to get there. In practical terms, that means you might never get there, since evolution can’t plan ahead [2].

On the other other hand…

1. This study considered only one function and only one environment. We have no idea how the look of the landscape would change if an experiment took into account that a primordial RNA molecule might have to do many jobs to “survive”, and it might “live” in an environment full of other molecules, ions, changing temperatures, whatever. (That would be a hell of an experiment. I think I might spontaneously explode into fireworks if someone did it.)
2. It’s not like this is really a problem from a plausibility perspective. The early earth did have a fair amount of time and potentially, quite a lot of RNA on its hands. I don’t think it originally would have had much longer RNA molecules than the ones in this experiment, not until RNA figured out how to make more of itself, but I’m pretty sure it had more than enough to explore sequence space.

4^24 molecules is about 2.8 x 10^14, or about half a nanomole (one mole is 6 x 10^23 molecules). One mole of 24-nt single-stranded RNA is roughly 8.5 kilos – I’d think you can fit quite a bit more than a billionth of that onto an entire planet with lots of places conducive to RNA synthesis. So I see no need to panic about the plausibility of random prebiotic RNA molecules performing useful (in origin-of-life terms) functions. (My first thought when I read this paper was “oh my god, creationism fodder,” but on closer inspection, you’d have to be pretty mathematically challenged to see it as such.)

So, in the end… I think I’ll settle for *SQUEEE!* After all, this is a truly fascinating experiment that doesn’t end up killing my beloved RNA world. On the question of replaying the tape, I’m not committed either way, but I am intrigued by anything that offers an insight. And this paper does – within its limited scope, it comes down on the side of evolution being very dependent on accidents of history.

Yeah. What’s not to like?

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Notes:

[1] I’ve worked a bit with RNA, and I have nothing but admiration for folks who do it all the time. The damned molecule is a total, fickle, unstable pain in the arse. And literally everything is full of almost unkillable enzymes that eat it just to mock your efforts. Or maybe I just really suck at molecular biology.

[2] I must point out that deleterious mutations aren’t always obstacles for evolution. They can contribute quite significantly to adaptation or even brand new functions. I’m racking my brain for studies of real living things related to this issue, but all I can find at the moment is the amazing Richard Lenski and co’s experiments with digital organisms, so Lenski et al. (2003)  and Covert et al. (2013) will have to do for citations.

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References:

Covert AW et al. (2013) Experiments on the role of deleterious mutations as stepping stones in adaptive evolution. PNAS 110:E3171-3178

Jiménez JI et al. (2013) Comprehensive experimental fitness landscape and evolutionary network for small RNA. PNAS advance online publication, 26/08/2013, doi: 10.1073/pnas.1307604110

Lenski RE et al. (2003) The evolutionary origin of complex features. Nature 423:139-144

This is when *I* need a good science blogger

Today you get to meet yet another of my random interests: the origin of life. (Is there a person with an interest in living things who isn’t fascinated by the origin of life?) And, since we sciencey types are very anxious about personal biases, I might as well start with a confession.

I love the RNA world hypothesis.

It was just one of those things that you learn at school/uni (I think it was 1st year molecular biology for me), and it’s so neat and elegant and compelling that you immediately fall in love. Sure, later, when you’re out of the inevitable simplicity of class, you learn about the nuances. The difficulties. But the evidence for still seems so convincing that you have no doubt that we’ll eventually solve the problems.

In case you aren’t familiar with it, the RNA world hypothesis is the leading solution to the chicken and egg problem that is the “central dogma” of molecular biology (diagram from Wikipedia):

DNA is great genetic hardware, but it’s nothing without proteins. Proteins are encoded in DNA, but the code is useless without proteins to read it. Making DNA requires proteins. But the proteins come from the DNA code. You see where this is going…

RNA takes the stage

The RNA world is an ingenious idea that elevates RNA from being merely the messenger between DNA and protein to centre stage. While its big brother DNA is a fairly stable and inert molecule, RNA is much more chemically active. It doesn’t like languishing in long, stable double helices – rather, it folds up into all kinds of odd shapes that can, surprisingly, catalyse a variety of chemical reactions. Just like proteins. Yet the “letters” of RNA can form complementary pairs, allowing for faithful copying. Just like DNA.

And, so the theory goes, there was a time when RNA was both the genome and the enzymes (enzymes made of RNA are called ribozymes). The right sort of RNA molecule could have copied itself without proteins [1], and performed whatever chemistry a primitive life form needed – also without proteins. Crucially, the right sort of RNA molecule could have invented proteins [2].

One of the key revelations to lend support to the RNA world hypothesis is that proteins in cells today are still made by RNA. Proteins are manufactured in ribosomes. A modern ribosome is a very complicated structure made of several folded-up RNA molecules and dozens of proteins. However, investigations of its structure (see Cech [2000] for a quick review) revealed that the place where amino acids are joined into a protein chain is all RNA – the proteins may support the RNA, but it seems to be the RNA that actually does the job.

Beautiful hypothesis vs. ugly facts?

So, everything is shiny and awesome and exciting. Ribozymes capable of all sorts of interesting chemistry [3] abound, and we have some very neat ideas regarding how RNA paved the way towards the modern protein-and-DNA world [2].

And then Harish and Caetano-Anollés (2012) come along, and I don’t know what to think.

A large part of the problem is that their methods go way over my head. I get the gist of their message. They figured out the relative ages of the RNA and protein components of the ribosome. The protein-synthesis parts – RNA and protein alike – turned out relatively new. They also found that the oldest protein parts interact with the oldest RNA parts – and seem to have coevolved. That, they say, would suggest that RNA and fairly large pieces of protein had a common history together before the future ribosome became capable of making proteins.

Yes, that means either that RNA didn’t invent proteins, or at the very least, that the “inventor” was not a precursor of the ribosome.

I really really don’t want to believe the former, and the latter possibility is a butchery of Occam’s razor without further evidence. But what else is left, if the study is correct?

One part of their results that I found intriguing is the structural similarity of the most ancient parts of ribosomal RNA to – you’d never guess – lab-evolved RNA-copying ribozymes. That is… oh, I don’t really know what it is, aside from “fascinating”. Did the ribosome start out as replication machinery, and turn into a protein factory only later? Or are the structures similar because reading the primitive genetic code required the same sort of molecular machine as copying RNA? Or is it even just coincidence?

And this is why I need a good science blogger. I need someone who deeply understands the paper and can translate it into something I can digest. Because at the moment, I can’t make heads or tails of this. I’m rather attached to the RNA world; it makes sense to me, and as far as scientific hypotheses go, it’s simply beautiful. Yet I can’t point to any obviously bullshit reasoning in the new study, other than where they seem to imply that because modern ribosomes need proteins to work, proteins must have been present in the ribosome from the start. (Which is a bit like every damn irreducible complexity argument advanced by creationists.) I just don’t have a good enough grasp on the methodology to tell whether it’s all solid or whether any of it is dodgy. Words fail to express how much that bugs me.

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Notes:

[1] Lincoln and Joyce (2009) and Wochner et al. (2011) came tantalisingly close to making/evolving the right sort of RNA molecule in the lab. The former’s pair of ribozymes can only copy each other by stitching together two half-ribozymes, but they can keep going at it forever and ever. Wochner et al.’s molecule can copy RNA using single letters as ingredients, but it runs out of steam after 90 or so of them. That’s several times better than the previous record, but still not long enough for the ribozyme to replicate its twice-as-long self.

[2] This excellent video describes one way it could have happened. When it comes to science education, cdk007 never fails to deliver!

[3] Including attaching amino acids to other RNA molecules (Turk et al., 2010) – look up tRNA if you don’t see why this is exciting 😉

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References:

Cech TR (2000) The ribosome is a ribozyme. Science 289:878-879

Harish A & Caetano-Anollés G (2012) Ribosomal history reveals origins of modern protein synthesis. PLoS ONE 7:e32776

Lincoln TA & Joyce GF (2009) Self-sustained replication of an RNA enzyme. Science 323:1229-1232

Turk RM et al. (2010) Multiple translational products from a five-nucleotide ribozyme. PNAS 107:4585-4589

Wochner A et al. (2011) Ribozyme-catalyzed transcription of an active ribozyme. Science 332:209-212