We heard in “The Organism’s Story” that living activity has a certain future-oriented (“purposive” or “intentional” or end-directed) character that is missed by causal explanations of the usual physical and chemical sort. This is true whether the end being sought is the perfection of adult form through development, or the taking of a prey animal for food.
An animal’s end-directed activity may, of course, be very far from what we humans know as conscious aiming at a goal. But all such activity nevertheless displays certain common features distinguishing it from inanimate proceedings: it tends to be persistent, so that it is resumed again and again after being blocked; it likewise tends to be adaptable, changing strategy in the face of altered circumstances; and the entire activity ceases once the end is achieved.
This flexible directedness — this interwoven play of diverse ends and means within an overall living unity — is what gives the organism’s life its peculiar sort of multi-threaded, narrative coherence. Life becomes a story. Events occur, not merely from physical necessity, but because they hold significance for an organism whose life is a distinctive pattern of significances.
The idea of narrative coherence, like the related idea of a governing context (Chapter 7), is a mystery for all attempts at purely physical explanation. This is why even the explicit acknowledgment of an organism’s striving for life — central as it may be for evolutionary theory — is discouraged whenever biologists are describing organisms themselves. It sounds too much as if one were invoking inner, or soul, qualities rather than material causes — acknowledging a being rather than a thing. And it is true that our physical laws, however combined, nowhere touch the idea of striving.
Biologists much prefer to identify single, definitive causes. The cell nucleus with its genome has long been viewed as the seat of such causation. But, as we saw in our discussion of epigenetics, the single-minded pursuit of genetic causes has forcibly redirected our attention to epigenetics, where we have discovered that genes are circumscribed and given their meaning by the narrative life of the entire cell and organism.
In what follows below we will consider this narrative coherence in a more detailed way — first, in relation to one of the many activities of the cell that can be considered under the heading of “epigenetics”. Then we will look more briefly at a startling phenomenon that, already on its face, renders absurd the idea of central genetic control. In both cases we will be focused on molecular-level activity, which is precisely where we have been most strictly trained to expect the absence of any coherence other than that of “blind mechanism”.
The discovery of RNA splicing in the late 1970s was one of the transforming moments in the history of molecular biology. To put it in informal terms: the cleanly autocratic mastery of DNA gave way to massive presumption by various scruffy elements of the cellular “rabble”. The idea had originally been that a molecule of messenger RNA (mRNA) was produced as a direct image of the “instructions” in a protein-coding gene and was then exported from the cell nucleus to the cytoplasm. There it yielded passively to translation, a process whereby a protein was supposedly produced according to the exact specifications of the “genetic code” previously copied from DNA into the mRNA.
Our growing knowledge of RNA splicing has, together with many other developments in molecular biology, exploded just about every aspect of this picture. We now know that, via an elaborately orchestrated improvisational drama, many so-called epigenetic elements in the cell (Chapter 8) come together to help decide what use will be made of any particular gene.
In particular, the cell has innumerable ways to obtain, design, and sculpt whatever proteins it needs at the moment. RNA splicing is just one of these — a massive reconfiguration process whereby a cell decides which portions of an initially produced (precursor) RNA to cast aside for other uses, and which ones to “splice” together into a mature mRNA. As we have come to expect by now, these choices are strongly context-dependent, with different protein variants being produced in different kinds of cell or tissue, or under different cellular conditions.
This splicing involves much more than a minor stitch or two. The large human dystrophin gene (whose malfunction is related to some forms of muscular dystrophy) is said to require 16 hours for its transcription from DNA into RNA. Of this time, 15 hours and 54 minutes is required for transcripton of the non-protein-coding RNA sequences that will have to be spliced out of the RNA in order to obtain a mature messenger RNA. That may be a somewhat extreme case, but it remains true that the sequences to be discarded are “commonly orders of magnitude longer” than the remaining portions fit for the synthesis of protein (Papasaikas and Valcárcel 2016).
But the most dramatic transformation involves the sequences remaining after removal of the non-protein-related (“noncoding”) content. The splicing activity can often select from among these sequences in differing ways, thereby determining which functional portions of the precursor molecule will be included in the mature mRNA. The protein eventually resulting will vary depending on these alternative splicing decisions. (The protein variations are referred to as isoforms.)
Over 90 percent of mammalian genes are thought to be alternatively spliced, contributing greatly to physiological complexity. According to one paper, “As cells differentiate and respond to stimuli in the human body, over one million different proteins are likely to be produced from less than 25,000 genes” (de Almeida and Carmo-Fonseca 2012).
Further, “even relatively modest changes in alternative splicing can have dramatic consequences, including altered cellular responses, cell death, and uncontrolled proliferation that can lead to disease” (Luco and Misteli 2011). The title of one technical paper makes the point vividly: “Cell Death or Survival Promoted by Alternative Isoforms of [the protein] ErbB4”.
You have doubtless heard many times how a mutation or engineered alteration of such-and-such a gene “causes” this or that result. How often, by contrast, do you hear that a slight change in the way your cells orchestrate the sculpting of this or that protein can make the difference between life and death?
The central player in the splicing drama is known as the spliceosome, which is not so much a rigidly fixed thing or structure as it is a complex performance. The performers include a few critically important small RNAs and over 150 proteins.1 Together — although in several, separate, coordinated groups that must continually reconfigure themselves during the process — they excise the protein-unrelated pieces of the RNA and then stitch together a selection of the ones remaining. Misjudging any of the potentially many places to cut the mRNA — shifting the point of severance by a single “letter”, or nucleotide base, out of (in many cases) thousands — could well render the resulting mRNA useless for producing protein, if not downright harmful.
We heard a little bit in Chapter 3, “What Brings Our Genome Alive?”, about the puzzle of topoisomerases. In a way that is difficult to fathom, these molecules make cuts in the DNA double helix in order to release knots and “untangle” the seemingly indecipherable spatial complexity of chromosomes (46 in the human case) that are tightly packed into the cell nucleus. But the challenge for the spliceosome as it does its work seems no less daunting. And the fact that there is indeed coherently describable work to do already takes us beyond normal physical explanation to the idea of an unfolding story.
The key, chemically active part of the spliceosome complex “is short lived and reconstructed from individual pieces for each splicing event”. (Papasaikas and Valcárcel 2016). This is the part that actually cuts and stitches together the RNA once the end-points for the next excision are chosen. Moreover, few of the scores of proteins required for the activity stay together throughout the intricate work on a single RNA. “At all transitions in the splicing process, the spliceosome’s underlying RNA-protein interaction network is compositionally and conformationally remodeled and at each step there is a massive exchange of proteins” (Wahl and Lührmann 2015).
But it gets worse. In multicellular organisms the mRNA being remodeled possesses particular sequences that are supposed to act as signposts for “attracting” the elements of the spliceosome to the correct sites for cutting and stitching. But these signposts are often ambiguous or contradictory, and provide only more or less vague hints. This is despite the extraordinary complexity of the task facing the spliceosome, and the large number of segments that commonly require removal.
“It has been proposed” [write two researchers] “that thousands of different sequences” can function as directives for the spliceosome, with only a few of the key “letters” in those sequences even being reliably present. Further, many sequences that look rather like splice sites are ignored by the spliceosome, while other sequences, despite lying at a distance from the splice sites, nevertheless contextually influence site recognition. So it appears that “hundreds of regulatory motifs may need to be integrated” (and understood) in order for the spliceosome to accomplish its surgery in harmony with current cellular needs (Papasaikas and Valcárcel 2016).
Using the thing-oriented (rather than process-oriented) language available to us, it is difficult not to speak of the spliceosome as a fixed structure, and equally difficult to avoid suggesting that it has a specific and well-defined task. What we see, however, is a remarkable plasticity. This is illustrated, for example, by the fact that “nearly all ‘activators’ of splicing can, in some cases, function as repressors, and nearly all ‘repressors’ have been shown to function as activators … it is clear that context affects function” (Nilsen and Graveley 2010).
But this context-sensitivity extends also to the very definition of the entire task, which looks utterly different, and requires wholly different approaches and capabilities on the part of the spliceosome, depending on the situation. Is the task to skip the next protein-coding segment of the RNA? Is it to make sure that a choice is made between two such segments — to retain only one and remove only one? Is it to choose an alternative location for the beginning or end of a particular segment? Is it, in at least some cases, to make the radical choice of preserving a non-protein-coding segment in the final mRNA?
Each of these operations demands a different sort of coordination among the many molecules involved, and the ways of approaching the work can vary, one might almost say, “wildly”. “Mechanisms of alternative splicing are highly variable, and new examples are constantly being found.”2 So there is not just one “spliceosome machine” (as some would like to call it), and not just one task. The cloud of molecules participating (or capable of participating, but “electing” not to) in the various splicing operations face the challenge of working together in an unimaginably sophisticated manner that somehow reflects the wider context and the needs of the cell.
Who will disagree with the researchers who write, in what might even be an understatement: “Working in a highly orchestrated manner, [the many parts of the spliceosome] perform incredible feats of molecular gymnastics with each round of splicing” (Chen and Moore).
The problem of what it actually means to say that “molecules do the work of splicing” is one of those vast blanks in scientific understanding that are easily papered over today in favor of informational generalities and convenient pictures of tiny machines busily, and in a “mechanistically” respectable fashion, carrying on the work of a cellular factory.
We already heard about the essential problem from cell biologist Paul Weiss (Chapter 7), who spoke about the many degrees of freedom possessed by the cell’s constituents in their watery medium, and about how these degrees of freedom are so remarkably constrained and disciplined toward intricate biological order at higher levels of observation. But it would be well to consider the issues in a specific and more detailed context, such as that of RNA splicing — and also to notice the general avoidance of these same issues within the biological community.
“Avoidance” seems the right term, for the problem is, in a way, widely recognized, although not meaningfully engaged. Referring to the “huge number of potentially regulatory elements in a very crowded nucleus”, University of Massachusetts geneticist Job Dekker wonders, “How do cells ensure that genes only respond to the right regulatory elements while ignoring the hundreds of thousands of others?” (Dekker 2013).
It’s a good and obvious question. An editor of Science amplifies it in a slightly different context this way: “If you think air traffic controllers have a tough job guiding planes into major airports or across a crowded continental airspace, consider the challenge facing a human cell trying to position its proteins”. A given cell, he notes, may make more than 10,000 different proteins under any particular set of conditions, and it typically contains more than a billion individual protein molecules at any one time. “Somehow, a cell must get all its proteins to their correct destinations — and equally important, keep these molecules out of the wrong places” (Travis 2011).
And once more: after a study showed that 70 percent of mRNAs in a cell are specifically localized, Robert Singer of Albert Einstein College of Medicine in New York City called it a “staggeringly large number”. He went on: “It’s almost as if every mRNA coming out of the nucleus knows where it’s going.”3
The entire problem is perhaps most vividly framed when we consider one further fact about RNA splicing. Not only is the spliceosome “a remarkably dynamic and flexible molecular machine; its transitions are so malleable that the whole reaction can eventually be reversed to generate precursor mRNA from spliced products” (Papasaikas 2016). More particularly:
Rather than being the one-way pathway typically drawn in textbooks, almost every step in the spliceosome cycle is readily reversible … [For example, regarding the first and second chemical steps in splicing,] not only can the spliceosome catalyze both chemical steps in forward and reverse, it can even convert spliced products … back into unspliced precursor mRNA! (Chen and Moore)
That is, the splicing choreography can take an already spliced RNA along with a section previously removed from it, and reinsert that section into the RNA.
The reversibility and flexibility underlying the finely gauged, discriminating, and “perceptive”4 activity of RNA splicing are hard to overestimate. It’s not just that “every major subcomplex addition step along the yeast spliceosome assembly pathway” has been shown to be reversible.5 Beyond this, many of the individual proteins coming together in the spliceosome (or remaining apart) are themselves subject to modifications that are often decisive for how the proteins will function within their current context.
And these modifications, too, are dynamic and reversible. They are also mutually entangled, with one kind of modification in one protein likely affecting, or being affected by, diverse modifications in other proteins. The untraceable lines of cause and effect blur into — and become subordinate to — the overall storyline.6
Everything I have said about RNA splicing testifies to a balance of conformations and movements that have the potential to tell any number of greatly or slightly differing stories, depending on the cellular context. But this is not a truth that many are eager to acknowledge. Instead, the topic gets changed. Job Dekker, who asked above how genes recognize and respond only to a minuscule proportion of the available regulatory stimuli, immediately went on to offer what he thought was one part of the answer:
Recent work has revealed a surprisingly simple strategy for matching genes to only some regulatory elements, which involves the spatial organization and folding of chromosomes inside the nucleus.
Certainly this folding, which we encountered in Chapter 3, is an important part of the answer — the answer, that is, to a different question: “What are the physical and chemical processes at work underneath the storyline?” But the question about the narrative thread itself is only broadened by this answer. Now we need to know: How does the extraordinarily intricate spatial coordination of events in the nucleus, including chromosome folding and the taming of “tangles”, occur in just the right way? What guides all the molecules involved in chromosome dynamics to manage the complex folding and spatial distribution of chromosomes so as to connect all the “right” loci to each other according to needs that may continually change during development and under varying conditions?
Dekker concludes his statement with this: “Future studies will no doubt unveil how [certain chromosome domains] are established and how they insulate genes from the wrong crowd.” Yes, and those studies will continue to reveal the explosively expanding universe of interweaving regulatory factors that only make even more remarkable the integral and unified, narrative performance of a single cell.
Despite Dekker’s choice of language, we can be sure that individual molecules do not “know” right from wrong — and he is surely not saying as much. But the language he so naturally feels pressured into using does serve to keep the real problem in view: cells and organisms do somehow work to avoid or correct errors and to achieve the right outcomes. But this is also to say: the explanatory task that researchers are supposedly addressing — How does the cell manage to accomplish what it does? — is not yet being touched by the enumeration of physical interactions that know nothing of right and wrong.
The central question of today’s biology is being ducked.
Our second case is a long way from RNA splicing — and also, it might seem at first, from the human being.
A dose of ionizing radiation equal to 10 grays (a measure of absorbed radiation) is lethal to the human body. Most bacteria cannot survive 200 grays. But then there is the bacterium known as Deinococcus radiodurans: it can endure over 17,000 grays and do quite well, thank you. Never mind that its genome is thoroughly shattered by the assault.
Here’s what happens. Ionizing radiation can damage DNA in various ways, perhaps worst of all by causing double-strand breaks. These are breaks across both strands of the double helix. The familiar bacterium, E. coli, not at all untypically, dies when it suffers about four double-strand breaks per each of its four-to-eight circular DNA molecules. Deinococcus radiodurans, by contrast, can survive over a thousand double-strand breaks. This means that it continues life after its genome is broken into many hundreds of small fragments. It does so by proceeding to put its genome back together again when living conditions improve — a daunting task, to say the least.
Deinococcus radiodurans is one of a small class of single-celled organisms with extreme radiation tolerance. Actually, it tolerates various other extreme conditions as well — some of which, such as dessication, likewise reduce its DNA to genomic shards. It can, for example, survive in a waterless desert for years until moistened again — which could happen, for example, when winds lift it in a cloud of dust from the Sahara, high into the atmosphere (where it is exposed to damaging ultraviolet radiation 100 to 1000 times that on earth’s surface), and across the Atlantic ocean to the South American jungles. D. radiodurans can be found on Antarctic ice, on dry frozen marble, and in the farthest depths of the sea.
Biologists have been intrigued by this peculiar survivor (along with some of its kin) for several decades, and of late they have clarified its story considerably. A central feature of that story is striking, because it points toward a truth about organisms in general, not merely those with extreme survival capabilities. The key finding is this: damage to DNA is not, in the most direct sense, what proves lethal about radiation. The primary issue, instead, is damage to proteins. As long as its proteins remain functional, a cell can reassemble even a badly fractured genome; but with damaged proteins, a cell is done for, with or without an intact genome.
D. radiodurans employs a number of strategies for preserving its rather commonplace “proteome”, or total inventory of proteins. These strategies include (1) preventing the oxidative damage that results from radiation, a goal it achieves in good part by means of an especially rich supply of antioxidants; (2) eliminating, before they can cause mischief, any proteins that do get damaged, while recycling their constituents; (3) scavenging amino acids and peptides (protein constituents) from the local environment, a capability that, together with the recycling, supports (4) newly synthesizing any proteins that need replenishing.
The proteome thus preserved is then able to go about the task of reconstructing a shattered genome — a task whose complexity at the molecular level is stunning. (Many a bright but befuddled graduate student has twisted his imagination into knots while trying to picture the actual textbook sequence of DNA damage repair in human cells.) Nevertheless, the task is accomplished in the cells of all organisms. What distinguishes D. radiodurans is its ability to carry out this task to an exceptional degree by maintaining its store of proteins intact under extreme duress.
In sum, according to Anita Krisko and Miroslav Radman, researchers at the Mediterranean Institute for Life Sciences who have been studying D. radiodurans, “biological responses to genomic insults depend primarily on the integrity of the proteome … This conclusion is the consequence of the fact that dedicated proteins repair DNA, and not vice versa”. Moreover, “this paradigm is fundamental in its obviousness (no living cell can function correctly with an oxidized proteome) and, if it is true, must be universal, that is, hold also for human cells”.
All this says something powerful about the longstanding genocentric (gene-centered) bias of biologists. Krisko and Radman delicately hint at the issue when they write in their paper:
The science of molecular biology was dominated by the notion of information, its storage, transmission, and evolution as encrypted in the nucleotide sequence of nucleic acids. But the biological information is relevant to life only to the extent of its translation into useful biological functions performed, directly or indirectly, by proteins. (Krisko and Radman 2013)
This truth, as they also point out, applies to our understanding of cancer and its treatment, which have long been focused on DNA abnormalities. But instead, “an effective cancer therapy by tumor cell killing should target the proteome, or both the proteome and genome, rather than the genome alone”. Which is almost to say: it should reckon with the coherent life narrative of the organism as a whole.
The work on D. radiodurans can remind us that the activity of an organism always reflects something like what we can only refer to metaphorically as a “sense of the whole”. Repeating what was said above: surely the protein molecules in this bacterium do not “know” what their “goal” should be in dealing with all those disordered snippets of DNA. But if the overall living context (Chapter 7) remains sufficiently intact, then the mysterious power of self-realization that we have been gently stalking in these several chapters — the coherent storyline of a life — continues to assert itself. The narrative, whatever its unexpected twists and turns, remains unbroken. If parts can be more fully constituted from their shattered fragments, it is because a functioning whole, with its intelligence, was already there.
The information we conceive as statically encoded in DNA is a kind of bland reduction of the living intelligence at work in cellular processes. When we occupy ourselves one-sidedly with genocentric information, it is (to employ a rough analogy) as if we elevated a notebook containing selected words, phrases, definitions, and grammatical guidelines to a pinnacle high above Moby Dick or Faust or War and Peace, worshipping the former as “information” while ignoring the informed and meaningful activity through which inert words and phrases are woven into soul-stirring tales.
A phrase-book or dictionary can be an essential resource, but it is the organism (Deinococcus radiodurans in the case we have been considering) that uses the dictionary to weave its own story — and even reconstructs the dictionary when the pages fall into a disorganized heap on the floor.
1. Estimates of the number of proteins participating in the spliceosome vary widely. Some have said there are more than 300, and others “only” 80 — a good indication of a fluidity of structure that is hard to nail down.
2. Wikipedia article, “Alternative Splicing”, accessed May 11, 2019.
3. Quoted in Travis 2011.
4. Obviously, I am not referring to our own conscious perceptive capacities. But neither am I referring to something less effective than our power of perception. Whatever brings the biologically coherent and needful results out of the currently inconceivable, creative “chaos” of the cellular plasm is far beyond our efforts to follow, let alone to reproduce. We have to think of a capacity higher than anything we consciously possess, even if — as the psychosomatic unity of the organism suggests — our consciousness is somehow contiguous with this higher capacity.
5. Chen and Moore 2014. Chemical reactions are in general reversible. But the authors here are noting that these particular reactions of the spliceosome occur under conditions where they are “readily” reversible.
6. There are many other aspects of RNA splicing not considered here — for example, the role played by certain metal ions in the shift between different protein conformations (and therefore between different protein functioning). Such ions are a long way from the macromolecules in which biologists normally invest their sense of cellular information, and yet their well-informed role is crucial to cellular activity.
Chen, Weijun and Melissa J. Moore (2014). “The Spliceosome: Disorder and Dynamics Defined”, Current Opinion in Structural Biology vol. 24, pp. 141-9. doi:10.1016/j.sbi.2014.01.009
de Almeida, Sérgio F. and Maria Carmo-Fonseca (2012). “Design Principles of Interconnections between Chromatin and pre-mRNA Splicing”, Trends in Biochemical Sciences vol. 37, no 6 (June), pp. 248–53. doi:10.1016/j.tibs.2012.02.002
Dekker, Job, Joanna Wysocka, Iain Mattaj et al. (2013a). “Nuclear Biology: What’s Been Most Surprising?”, Cell vol. 152 (March 14), pp. 1207-8. doi.org/10.1016/j.cell.2013.02.041
Graveley, Brenton R. (2011a). “Splicing Up Pluripotency”, Cell vol. 147 (Sep. 30), pp. 22-4. doi:10.1016/j.cell.2011.09.004
Krisko, Anita and Miroslav Radman (2013a). “Biology of Extreme Radiation Resistance: The Way of Deinococcus radiodurans”, Cold Spring Harbor Perspectives in Biology 2013;5:a012765. doi:10.1101/cshperspect.a012765
Luco, Reini F. and Tom Misteli (2011). “More Than a Splicing Code: Integrating the Role of RNA, Chromatin and Non-coding RNA in Alternative Splicing Regulation”, Current Opinion in Genetics and Development vol. 21, pp. 366-72. doi:10.1016/j.gde.2011.03.004
Nilsen, Timothy W. and Brenton R. Graveley (2010). “Expansion of the Eukaryotic Proteome by Alternative Splicing”, Nature vol. 463 (Jan. 28), pp. 457-63. doi:10.1038/nature08909
Papasaikas, Panagiotis and Juan Valcárcel (2016). “The Spliceosome: The Ultimate RNA Chaperone and Sculptor”, Trends in Biochemical Sciences vol. 41, no. 1 (Jan.), pp. 33-45. doi:10.1016/j.tibs.2015.11.003
Travis, John (2011). “How Does the Cell Position Its proteins?” Science vol. 334 (Nov. 25), pp. 1048-9. doi:10.1126/science.334.6059.1048
Wahl, Markus C and Reinhard Lührmann (2015). “SnapShot: Spliceosome Dynamics I”, *Cell*, vol. 161, no. 6, (June 4), pp. 1474-1474.e1. doi.org/10.1016/j.cell.2015.05.050
Steve Talbott :: The Mystery of an Unexpected Coherence