Evolutionary Elaboration of mRNA Splicing

An RNA helicase scoots through the spliceosome to advance the process of mRNA splicing. And some other tricks.

In our cells, virtually all mRNAs transcribed from DNA have to go through an editing process to cut out intervening junk called introns. This process is called splicing, and its evolutionary origin, later elaborations, and current mechanism are all quite interesting. Life didn't start with introns, and only eukaryotes have them as a regular feature of their genomes. They appear to have arrived with the bacterium that became our mitochondria, which come from a lineage that has (relatively few of) what are called group II self-splicing introns. These are RNA segments that behave a bit like transposons, being able to jump into DNA, and then reverse-transcribe that segment into a copy of itself in genomic DNA. 

The ur-eukaryote seems to have had an incredibly prolific infection, which left its host genome riddled with these bits of DNA. A key point is that, in group II introns, their splicing out of a transcribed RNA message is auto-catalytic- entirely mediated by their own RNA structure. They are self-propagating parasites, which have, over time in eukaryotes, been tamed to become fertile aspects of our own gene regulation and evolution. For example, introns often fall between protein domains, allowing these relatively compact modules of protein structure to be replicated, moved, and plugged, via rare mutational events, into new settings to contribute new functions to existing or novel proteins.

A map of a group II self-splicing intron. In red are the ends of the host RNA (or DNA) which are to be either jumped into or excised out of. The rest is the structure of the intron, which carries its own catalytic ability to do these reactions. This kind of thing is what appears to have turned into our own splicing and intron/exon systems, since the core catalytic mechanisms, such as the use of lariats and branch points and RNA catalysis, are the same.

Representation of the core spliceosomal reactions in eukaryotes, which result in a free lariat form of the excised intron, and the joined exons, which go off to code for their protein. "SS" stands for splice site. The sequences in red characterize introns.

The mechanism of intron excision in our cells is, at its core, still RNA-based, even though there are now also hundreds of proteins involved in the rather massive machinery of what is now called the spliceosome. It is clear that over evolutionary time, what was originally an unwelcome and shocking invasion of proto-mitochondrial introns into the proto-nuclear host genome has been regulated, speeded up, accessorized, and integrated into our normal method of gene expression. The spliceosome is the result- a huge and dynamic complex that uses key bits descended from the original RNA catalytic components to guide and catalyze the splicing reaction.

Representation of the core splicing reactions, with the key small RNAs added in (U1, U2, U4, U5, U6). These both guide (by direct RNA-RNA hybrid formation) and perform catalysis at the two chemical bond-breaking/reforming steps.

There are three key locations seized upon by the spliceosome. First is the 5 prime splice site- the end of the coding exon and beginning of the intron, typically a "G" nucleoside at the start of the intron. Second is the branch point, an "A" near the end of the intron, which is where the chemistry of splicing begins. And third is the 3 prime splice site- the end of the intron, with another "G" nucleoside, next to the beginning of the next coding exon. While the first two sites are specifically recognized by RNA components of the spliceosome, (U1 at the 5 prime splice site and U2 at the branch site), the 3 prime splice site is simply recognized by scanning for the first "AG" downstream of the branch site.

The first reaction is to bring the branch site and the 5 prime splice site in proximity, such that the branch site A covalently invades the G at that site and displaces it, releasing the exon end and forming a loop (called a lariat, in red above) in the intronic RNA. The second reaction is to bring the 5 prime exon end over to the 3 prime exon end, and similarly prompt and invasion that links them, displacing the intron entirely.

So simple to describe, but not so easy to do. Accuracy is paramount, since the three-codon reading frame of mRNA would be destroyed by even a 1 nucleoside error in splicing. Splicing now gates the export of mRNA from the nucleus, so that only fully and accurately spliced transcripts get out to the ribosomes in the cytoplasm for translation to protein. This gating has been considered by some the very reason that the nucleus exists at all- a way to solve some of the knotty problems that arose in very early eukaryotic evolution when all these introns invaded. 

Another reaction scheme of splicing, showing the key RNA and some other proteins along the way, principally the key helicases that help drive things forward. Note where PRP2/ATP comes into the picture, just as the complex is preparing for the first catalytic step.

Be that as it may, it is clear that the originally RNA-only mechanism changed over time by accreting proteins that each decided they had something useful to add to the process. At the same time, the RNA got separated into several pieces (on independent genes) that could then be carried and precisely manipulated by these helping proteins. The spliceosome now involves five distinct small RNAs and over 200 proteins, which engage in a complex ballet of sequential steps. A special class of these proteins, the helicases, are the subject of a recent paper that provides new structural information. Helicases are proteins that can use the power of ATP to unwind DNA or RNA, or just chug along it. At least eight such proteins participate in splicing. 

Structures from a recent paper, showing how PRP2 (at bottom, in violet) chugs its way along the mRNA intron (blue) into the very heart of the spliceosome complex, partially evicting the SF3B1 protein (green), among others, and prompting many other changes. At top is shown the 19 nucleoside stretch of the mRNA that was traversed, getting close to the branch site "A" in red. 

The paper makes the interesting observation that, structurally, most of the helicases reside on the periphery of the spliceosomal complexes, while the catalytic and guiding RNA are, naturally, at the center. They use a mutant form of one of them, Aquarius, to freeze spliceosomes in a key conformation just before the branch site and 5 prime splice sites are brought together. In combination with a bunch of other structural work by others in this and other labs, they show that one dynamic event is the tracking by a second helicase, PRP2 (violet, above), that brings it from its peripheral position (b, at bottom) along the intronic mRNA (blue strand) into the core of the splicesome near the U2 RNA (c; U2 is not shown here). They show that PRP2 traverses 19 nucleosides (top, a), a rather remarkable trip that forms part of the sequence of events that brings the branch site and 5 prime splice site close to each other.

Further structures, focusing on the catalytic site and RNAs. Note how the branch site (red, "BS-A") is, after the action of the Aquarius helicase, (third panel), brought in tightly close to the 5 prime splice site (green, "5'SS) in the C, or catalytic, complex. The U2 and U6 RNAs then have an easy job of bond-exchanging catalysis.

So it turns out that these helicases appear sometimes to be used as ratchets, that start on the outside of the complex. Once activated by some prior trigger, they pull on a thread in a way that helps to overall process forward. The progression of PRP2 into the spliceosome core evicts a bunch of other proteins and activates the other helicase Aquarius. That protein is likewise positioned perpipherally but is hanging onto another thread of the intronic RNA and helps to further push the branch and 5 prime splice sites together, in a way that finally leads to the desired reaction. Note in the image above that it is the RNAs that occupy the central reaction site- the intron in blue (green), and the U6 and U2 RNAs, which catalyze this first key reaction of splicing.

RNAs are not great catalysts, so it is understandable that, as in the case of translation by the ribosome, a bunch of proteins shoehorned their way into the process (over evolutionary time) in ways that evidently made splicing more accurate and more rapid. Indeed, yeast cells get along without the Aquarius protein at all, though they otherwise have a very similar splicing apparatus, showing that the accretion of proteins on the spliceosome did not end in very early stages of eukaryotic evolution, but continued through the origin of metazoans, and may still be continuing. The added proteins did this through using their talents for precise spatial positioning, and for the use of energy (from ATP) to drive things ahead, if only by intricate conformational ballet rather than direct catalysis.

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The Tragedy of Daniel Boone

Pathfinding and hunting his way through the paradise the Indians had built.

Daniel Boone is (or used to be) one of the most iconic / archetypal figures in US history and popular consciousness. His remains have been fought over, his life mythologized and serialized, and his legacy cherished as heroic and exemplary. It all began with his trusty rifle, with which he was the surest shot. He was a pathfinder, never lost in the vast wilderness he explored and helped settle. And he was a steadfast leader of men, rescuer of damsels in distress, and killer of Indians. What's not to admire? His definitive biography, by John Faragher, paints a more ambivalent picture, however.

Boone loved the woods- loved hunting, loved nature, and loved solitude. Given those talents and tendencies, he naturally strayed from the borderlands of North Carolina into the mountains, becoming a full time hunter and trapper. In a couple of early forays into what we now know as Kentucky, he hunted on a commercial basis, wasting the animals to pile up hundreds of pelts, which his employees / colleagues processed in camp. 

The biography emphasizes that what Boone found in Kentucky was a paradise- lush and full of game. The region, believe it or not, was full of not just deer and beaver, but bear and buffalo. It is the kind of eden that had been encountered by Europeans many times over in the "New World". Fisheries of unimaginable richness, skies full of birds, forests as far as the eye could see. Kentucky was not an uninhabited eden, however- it was the cherished hunting ground of native Cherokee and Shawnee, among others, who saw exactly what Boone saw, but responded to it differently. Not with plunder and destruction, but with care and stewardship.

Boone blindly shot away, and then followed his cultural programming further by leading his family and many others across the mountains to found Boonesborough, building a fort and defending it against numerous Indian attacks. The biography notes that Boone's parents had ten children, and he had ten children, and his children had similar sized families. One can imagine where that kind of reproduction leads, to desperate expansion and heedless use of resources. While acknowledged as the pioneer of Kentucky settlement, Boone was no businessman, and all his grasping for land in the speculative rush that developed in his wake came to naught. He was sloppy in his paperwork and was outlawyered and out-cheated at every turn. One may see the personality type of his adversary in the current senior senator from Kentucky, Mitch McConnell. Boone was all too honest and simple, having been raised a Quaker.

Portrayal of the siege of a stockade, not unlike that of Boonesborough, as Native Americans try to drive off the cloud of locusts denuding their land.

The game had been hunted out, the people had become unfriendly and dense underfoot, and Boone's property and business schemes had all fallen apart. In despair over what he had wrought in Kentucky, Boone pulled up stakes and moved out to the next frontier, near St. Louis. An extremely late hunting trip has him heading through what is now Yellowstone park, reliving for the last time the kind of eden that Native Americans had nurtured with their respect for the value and cycles of nature, and even more, with their light footprint as small populations.

European culture and immigrants have accomplished wonderful things in America. But decimating its natural wonders, resources, and native peoples is not one of them. Daniel Boone was caught up in the economics of inexorable population growth and the need to make a "business model" out of hunting and trapping. Well, what comes of that is not pretty, and not at all sustainable of what had brought him into the woods to start with.

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Why Does Wyoming Emit 57 Times as Much CO2 per Capita as California?

Diversity is not always a good thing. States are in very different places when it comes to the carbon intensity of electricity generation.

California has been working hard to become a more sustainable place, from an energy and emissions perspective. Compared to the baseline of 2000, population has risen 15%, electricity production has held steady, and emissions are down 15%. We have a very long way to go, but are diligently chipping away at the sustainability problem. Look across the country, however, and it is quite a different picture. A remarkable map shows each state's per capita emissions just for electrical power production.

Map of the US showing per capita CO2 emissions from electricity generation alone.

It turns out that most states emit multiple, some many multiple, times the atmospheric and climate pollution of states like California. It is clear that there is very weak regulation on the federal level, and that we have hardly progressed from the laissez-faire mine and drill policies of the last century. Aside from hydropower concentrations in the Northeast (Vermont, Maine) and Northwest (Washington, Idaho), no state has lower emissions than California, and as noted in the title, Wyoming puts out a whopping 57 times more carbon per person per year solely in the electrical sector.

Part of the problem is simple chemistry. Coal was the traditionally cheapest source of energy, but has high carbon emissions (not to mention sulfur, nitrogen, ash, and other noxious pollutants and waste). Its general formula is CH, with a roughly 1:1 ratio of carbon and hydrogen. In contrast, methane is CH4, with four hydrogens per carbon. Each of those bonds (whether carbon-carbon or carbon-hydrogen) yields the same amount of energy. So per unit of energy released, methane emits roughly half the CO2. On top of that come the costs of scrubbing out the sulfur dioxide and nitrogen compounds that have been the target of decades of policy against acid rain, which further decrease the efficiency of coal. Imagine on top of that the huge (about 1/3 of total yield) energy cost of scrubbing out the CO2 from coal smokestack emissions, compressing, and sequestering it underground, and one can appreciate what a pipe dream this "solution" is, to keep coal combustion a viable energy source.

Coal combustion is not, indeed, viable at all, if one gives any thought to the atmosphere or the rest of the environment. And it isn't even economically viable any more against renewable wind and solar energy. But there is so much sunk investment in the power plants and mines, that change is hard to motivate. In addition, there are the hidden costs of wind and solar, being the batteries, grids, and other backup power sources needed to cover their intermittent nature. Those are real concerns. But one of the biggest issues is simply the nature of regulation in various states. California has set up complex rules to make utilities sensitive to the cleanlines of their power mix, and their cost structure. Other states have simpler regulatory systems that give utilities markups for all capital expenditures and investments, allowing them to pass through such white elephants as wildly over-budget nuclear power plants. And also continue paying for coal that is costing more than renewable, competitive sources of energy.

More broadly, we need to keep raising the costs of pollution on a wide, federal basis, to a level that forces recalcitrant states to advance global climate goals with clean energy. The US still gets almost a quarter of its power from coal. Much of this is generated and exported from Wyoming, either as electricity or as coal trains. This needs to stop. Many of the most emitting states lie in the midwest and Rockies, which have outstanding wind energy resources. The energy is there for the taking, but it has become a political issue, with red states now dedicated to thumbing their noses at the libtard environmentalists, even if it means spiting their own faces in terms of total electricity costs, pervasive pollution, and appalling jobs.

Trends of fuel sources for electricity generation in the US. Coal has decreased steadily, but still comprises roughly a quarter of generation.

While renewable energy has made astonishing strides in efficiency and cost, it is clear (as we have learned in California) that carrots alone will not make the energy transition happen and save the environment. The government predicts, under current and foreseeable policies, that emissions from the electricity power sector will remain the same out to 2050. That is unacceptable. Sticks have to be used as well, to internalize the enormous and ever-growing costs of atmospheric pollution onto the sources of that pollution- the diverse and sometimes egregiously dirty fuels that feed our insatiable appetite for power.

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Damned if You do, Damned if You Don't

The Cherokee trail of tears, and the Palestinian conundrum.

History is a long and sad tale of conflict, interspersed with better times when people can put their animosities aside. Just as economics deals in scarcity and its various solutions, history likewise turns on our inevitable drive towards overpopulation, with resulting scarcity and conflict. Occasionally, special technological, spiritual, organizational achievements- or catastrophes- may allow periods of free population growth with its attendant bouyant mood of generosity. But more commonly, groups of people covet each other's resources and plot ways to get them. This was one of the lessons of Malthus and Darwin, who addressed the deeper causes of what we see as historical events.

The "New World" provided Europeans with an unprecedented release for their excess populations, especially the malcontented, the desperate, and the ambitious. They rhapsodized about the "virgin" lands that lay open, generally dismissing the numerous and well-organized natives present all over these lands, as "savages", occupying a lower technological and theological level of existence. There were plenty of rationalizations put forth, like Christianizing the natives, or "civilizing" them. But the hypocrisy of these formulations becomes clear when you consider the fate of the Cherokees, one of the "five civilized tribes". 

By the early 1800's, a couple of centuries of contact had already gone under the bridge, (as narrated by Pekka Hämäläinen in "Indigenous continent"), and native Americans were all integrated to various degrees in trading networks that brought them European goods like guns, pots, knives, and novel practices like horse riding. The Cherokees, occupying the lower Appalachians and piedmont between what is now Georgia and Alabama, were more integrated than most, adopting European farming, living, schooling, and governing practices. They even owned African American slaves, and wrote themselves a US-modeled constitution in 1827, in the script devised the scholar Sequoya.

Did this "progress" toward assimilation with the European culture help them? Far from! Their excellence in farming, literacy, and government raised fears of competition in the white colonists, and the Georgia state government lobbied relentlessly for their removal. Andrew Jackson finally obliged. He pressured the Cherokees to re-open their status as a settled nation, devised a removal treaty with a minority party, and then sent all the Cherokees in the region (about 16,000) off on the Trail of Tears, to the barren lands of Oklahoma. These Cherokees lost roughly a quarter of their population along the way, in a brutal winter. Compare this with the partition of India, where about twelve percent of the refugees are thought to have perished, out of roughly 16 million total.

A small part of the annals of ethnic cleansing, US edition. Needless to say, the "Indian territory" ended up a lot smaller than originally promised.

 

Georgia was thus ethnically cleansed, and does not seem to experience a great deal of regret about it. The logic of power is quite simple- the winner gets the land and spoils. The loser is lucky to not be killed. That the Europeans were significantly more powerful than their native antagonists doesn't change the logic, though it might appeal to our empathy and nostalgia in retrospect. The Cherokees and other Native Americans might have been accepted into US society. They might have been given one or two states for their sovereign governments, as the Mormons managed. There were a lot of possibilities that might have made us a more interesting and diverse nation. But at the same time, most Native Americans participated fully in the politics of power, terrorizing each other, making slaves of each other, and killing each other. They were not innocents. So the fact that they came up against a stronger power was hardly a novelty, though in this case that power was blundering and cruel, shared very few of their cultural coordinates, and was highly hypocritical about its own.

All this comes to mind when viewing the Israeli-Palestinian conflict. Israel won the major Middle East wars that so dramatically emasculated the Palestinians, first in the civil war that left Jordan and Egypt in charge of the Palestinian areas, then in the 1967 war that left all these areas in Israeli hands. But what to do with them? On founding, Israel was a liberal, New Testament kind of country, with humanist values and lefty kibbutzim. The then-recent Holocaust also caused a bit of hesitance when it came to either killing or exiling the losing Palestinians. Indeed, given that its neighbors Jordan and Egypt lost these wars, it would have made some sense at that time to deport all the Palestinians, of which there were about one to two million. But rather than do that, or make a firm border, Israel immediately started encroaching into Palestinian territory with security areas and "settlements", and has set up an ever more elaborate, though selectively porous and self-serving, security and boundary system.

Both sides have a schizophrenic reaction to the other. On the Palestinian side, the psychology of losing has meant quietism and acquiescence by some, but resentment and militantcy by others. Both lead to a spiral of worse treatment, the weakness of the former inviting abuse, and the desperate depredations of the latter inciting revenge, "security" measures, and tighter occupation. The provocations by each side are unendurable, and thus the situation deteriorates. Yet, in the end, Israel has all the power and the responsibility to come up with a long term solution. Over the decades, Israel has morphed from its founding ethos into something much more conservative and Old Testament, less beholden to the humanisitic ideals of the post-WW2 period. The wanton killing, starvation, and collective punishment of Gaza makes visible this moral breakdown.

The Palestinians can't win either way, either through Hamas's implacable hatred and impotent attacks, nor through the acquiescence of the Palestinian National Authority, which, in thanks for its good behavior, has received the creeping expansion of Israeli "settlements" on its land. These now take up, according to a detailed map, about 1/3 to 1/2 of the land of the West Bank. Overall, the options are: 1) to expel the Palestinians outright, which appears to be, for Gaza at least, where Israeli policy is heading, (made more ironic by the realization by historians that the Biblical Exodus never actually took place), or 2) to continue to muddle along in a torturous occupation with creeping dispossession, or 3) to grant Palestine some kind of autonomy and statehood. Assimilation, (4), long dreamt of by some, seems impossible for a state that is fundamentally an ethnic (or theological) state, and whose whole raison d'etre is ethnic separation, not to even mention the preferences of the Palestinians. Though perhaps assimiliation without voting rights, in sort of semi-slavery or apartheid, is something the Israelies would be attracted to? Perhaps insignia will need to be worn by all Palestinians, sewn to their clothing?

Map of the West Bank of the Jordan, color coded by Palestinian marginal control in brown, and settler/Israeli control in red.

What should happen? Indigenous Americans were infected, decimated, hunted down, translocated, re-educated, and confined to a small and very remote system of reservations. Hopefully we have have progressed a little since then, as a largely European civilization, which is putatively shared by Israel. Thus the only way forward, as is recognized by everyone outside Israel, is the two-state solution, including a re-organization of the Palestinian territories into a final, clearly demarked, and contiguous state. Israel's current political system will never get there. But we can help the process along in a few ways.

First, it is disappointing to see our current administration shipping arms to Israel at a furious pace, only to see them used to kill thousands of innocent, if highly resentful, civilians. Israel has plenty of its own money to buy whatever it needs elsewhere. We need to put some limitations on our military and other aid relationships, to motivate change. (Though that raises the question of Israel's increasingly cozy relationship with Russia). Second, we should recognize Palestine as a state, and bring forward its integration into the international system. This will not resolve its borders or myriad security and territory issues viz Israel, but it would helpfully motivate things in that direction. Israel has constantly cried wolf about the lack of a credible partner to negotiate with, but that is irrelevant. Israel is perfectly capable of building the walls it needs to keep Palestinians at bay. But then it wants pliant workers as well, and a peaceful neighbor, security viz Jordan and Egypt, territorial encroachments, and many other things that are either destructive, or need to be negotiated. 

By far the most constructive thing that could be done is to freeze and re-organize the Jewish settlements and other periphernalia that have metastasized all over the West Bank. There is no future without a decent and fair solution in territory, which is the third big thing we need to press- our own detailed territorial plan for Palestine. For one thing, Israel could easily vacate the whole corridor / valley facing Jordan. That would give a consolidated Palestine a working border with a country that is now peaceful, quite well run, and friendly to both sides. There are countless possible maps. We just need to decide on one that is reasonably fair and force it on both sides, which are each, still after all these years, apparently unwilling to imagine a true peace. This means principally forcing it on Israel, which has been the dominant and recalcitrant party the entire time.

The Cherokees are now one of the largest indigenous populations in the US, at roughly a quarter million, with their own territory of about seven thousand square miles in Oklahoma. They have internal and partial sovereignty, which means that they deal with their own affairs on a somewhat independent basis, but otherwise are largely subject to most laws of the enclosing governments. The Cherokees could easily have been assimilated into the US. Only racism stood in the way, in a mindset that had long descended into a blind and adversarial disregard of all native Americans as "others", (the irony!), competitive with and less than, the newly arrived immigrants. We could have done much better, and one would like to think that, a hundred or a hundred and fifty years on, we would have.

In the end, the West (read as European civilization, as developed out of the ashes of World War 2) is either for or against wars of aggression, ethnic cleansing, apartheid, and human rights. Israel has won its wars, but never faced up to its responsibilities to the conquered Palestinians, and has tried to have it both ways, to be viewed by the world as a modern, enlightened state, even as it occupies and slowly strangles the people it defeated decades ago. 

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