LncRNA: Goblins From the Genomic Junkyard

One more addition to the zoo of functional RNAs.

A major theme over the last couple of decades of molecular biology is the previously unanticipated occurrence of many sorts of small and not so small RNAs that do not code for proteins. In addition to buttressing the general proposition that RNA came early in the history of life and retains many roles beyond being merely the conveying medium of code from DNA to protein, these novel RNAs illuminate some of the complexity that went missing when the human genome came in at under 20,000 protein coding genes.

The current rough accounting of human genetic elements. While we have only 19,954 protein coding genes, we have a lot of other material, including almost as many pseudogenes, (dead copies of protein-coding genes), and even more RNA genes that do not code for proteins. The count of mRNA transcripts is high because splicing of a protein-coding gene is frequently variable and can generate numerous distinct mRNA messages from one gene. Similarly, the start and stop points of transcription can be variable for both mRNA and lncRNA genes.

RNAs perform many functions, such as the catalytic core of the ribosome, the amino-acid complementary-coding role of tRNAs, the catalytic core of the mRNA splicing apparatus, guides to edit and modify ribosomal RNAs, and miRNAs that repress expression of target genes. LncRNA stands for long non-coding RNAs, which were discovered by global analyses of RNA expression. Long transcripts were found that did not have protein coding frames, did not clearly derive from degraded pseudogenes (degraded copies of protein coding genes), and which occasionally still had significant conservation and thus evident selective constraint and function.

Another piece of background is that these expression analyses have found, as the technology advanced in sensitivity and comprehensiveness, that most of our genome is transcribed to RNA. Not only do we have a large amount of junk DNA, like transposons, repetitive elements, pseudogenes, intron and regulatory filler, etc., representing at least 90% of the genome, but most of this DNA is also transcribed at a low level. We have quality control mechanisms that dispose of most of this RNA, but there have been partisans of another perspective, particularly among those who first found all this transcription, that these transcription units are "functional", and thus should not be dismissed as "junk".

Eugene Koonin is not of that persuasion. His recent review of this field, and of lncRNAs in particular, with Alexander Palazzo, generates an extremely interesting model of why most of this is junk, and how such junk RNA can occasionally gain function. Some lncRNAs are important, typically helping nearby genes stay on. One of the most significant lncRNAs, however, represses its nearby genes, and is central to the process of X inactivation. XIST is 17,000 nucleotides long- very long for a non-coding RNA- and binds to dozens of proteins including chromatin remodeling enzymes and X-chromosome scaffold proteins, all in a byzantine process that shuts down the extra X chromosome that females have. This prevents the genes of that chromosome, which encompass many functions, not just sex-specific ones, from being expressed two-fold higher in females than in males.

How to make sense of all this? How can there be many thousands of lncRNAs, but only a few with function, and those functions rather miscellenous, typically local, and centered on transcriptional regulation? The tale begins with one of the many quality control features of the transcription apparatus. When a gene is transcribed, the polymerase as it goes by deposits chromatin marks (on the local histones) that prevent other transcription complexes from initiating within the gene. This prevents extra initiation events that would produce truncated proteins, which can sometimes be very harmful, lacking key regulatory domains. So the theory posits that much of the stray transcription of junk DNA through the rest of the genome, especially in the form of long lncRNAs, has a similarly repressive effect, reducing local initiation within those "gene" bounds. This might be particularly helpful to prevent interference with regulatory events happening in those regions, controlling transcription through the region rather than allowing it to happen sporadically all over.

As a first step, it is innocent enough, and not likely to have strong selection constraints, typically of a low level, and perhaps eventually responsive to some regulatory events, depending on the needs of the nearby coding gene. Nor would the lncRNA that is made have any function at all. It would be junk very literally, would not get spliced, or exported out of the nucleus, and probably get degraded promptly. Its sequence is under no particular selection, and would drift in neutral fashion. The second step then happens if this RNA were to gain some kind of function, such as binding some regulatory protein. There are many RNA and DNA binding proteins in the cell, so this is not difficult. Xist binds to over 80 different proteins. These proteins then might have local effects, as long as the lncRNA remains attached to its own transcription complex during its own synthesis. Such effects might be activating the nearby gene, loosening or tightening nearby chromatin. Given the (arbitrarily large) size of the lncRNA, and the typically small size of nucleic acid binding determinants that proteins recognize, there is little limit to how many such interactions could be accumulated over time, always subject to selection that likely centers around fine-tuning of effects on nearby genes. Indeed, this regulation could allow the relaxation or loss of more proximal regulators, making the lncRNA increasingly essential. After enough interactions accumulate, the lncRNA may remain tethered to local landmarks, and its activity persist after its synthesis ends, prompting selection against its degradation.

In this way, increasingly elaborate mechanisms can be built up, out of very modest selective effects, combined with a lot of drift and exploration of neutral mutational space. This theory provides a rationale for what we are seeing in the lncRNA landscape- a huge number with little to no ascertainable function, but a few that have grown into significant regulators of their local or extended chromatin landscape. It also informs the mechanism by which they function, not as some new exciting mechanism of action by a discrete RNA species, as was found with miRNAs, for instance, but rather an agglomeration of adventitious interactions that will be different in each case, and highly variable in effect.

"Indeed, although complexity in biology is generally regarded as evidence of “fine tuning” or “sophistication,” large biological conglomerates might be better interpreted as the consequences of runaway bureaucracy—as biological parallels of nonsensically complex Rube Goldberg machines that are over-engineered to perform a single task"

 

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Rise and Fall of the US

What happened to our 20th century solidarity?

A recent issue of The New Republic carried an article by its publisher that discussed Robert Putnam's diagnosis of the decline of American civic community and solidarity. In the generational arcs of US history, we have had high solidarity, and consequent productive and progressive political eras, only a few times- the colonial era, the Republican interlude while the South had seceded, the progressive era around the turn of the 20th century, and the post-WW2 boom. Perhaps much of the 20th century could be classified that way, up to the 1970s. At any rate, we are obviously not in such an era now. We are, in contrast, floundering in an era of incredible political and social divisiveness, of unproductive public institutions, and of social atomization.

"Just as Putman and Garrett identify an upswing, they also trace a decline beginning in the 1970s. For this, too, they offer an explanation that departs from the standard historical narrative, suggesting that it was not Ronald Reagan who brought the long period of liberal rule to an abrupt halt, but rather the baby boomer of the 1960s who, turning from the communitarian idealism of the early part of the decade toward a more self-oriented direction, set off a chain reaction that ended up blowing the whole Progressive-liberal order to smithereens."

But the article does not really articulate what happened, other than to cite the many dramas of that time, and propose that the US had a bit of a "nervous breakdown", in a transition from a conformist 50's, through the wide-open and tumultuous 60's, to the me-centered 70's. Perhaps this dates me, but I did live through some of those times, and I think can offer a more specific analysis. I'd suggest that the principal elements of the downturn arose from fundamental violations of trust by the state. The US had conducted WW2 with great moral and logistical authority. The grunts always grumbled, and there were plenty of fiascos along the way, but overall, there was a consensus that the elites and people in charge knew what they were doing. They not just won the war, but fostered unprecedented prosperity in its wake. 

All this turned around in the late 60's. I am also reading a history of the CIA, by Tim Weiner, "Legacy of Ashes". This is a deeply biased book, focusing on every failure of the CIA, pronouncing it as an institution utterly and irredeemably incompetent. What is noticeable, however, is that the CIA's successes are generally far more costly than its failures. The coups it sponsored in Iran and Guatemala, et al. came back to haunt us down to the present day. Eisenhower founded this pandora's box of disastrous meddling, (i.e. covert action) and Kennedy accelerated its use. One of its signature accomplishments was the slow process of getting us enmeshed in the Vietnam war. This was the single most influential disaster that discredited the US government to its own citizens. While in principle, we were doing a great thing- saving South Vietnam from communism and totalitarianism- in practice, we had no idea what we were doing, did not understand the nature of the civil war, or the impossible corruption of our allied government, and conducted the war in a fog of lies and delusions. The daily body counts were a visceral expression of revulsion against the state.

But this kind of incompetence became a pattern in major events like Watergate, inflation, the oil crisis, and the Iran hostage crisis. Each one showed that our leaders did not know what they were doing- the best and brightest turned out unequal to the crises we faced. A succession of presidents fell victim to fundamental breaches of trust with the country. Inflation, for example, made us feel helpless- that the money itself was being eaten away by processes that were virtually occult in their mystery and darkness. Gerald Ford urged a kind of vodoo economics- that perhaps a public relations campaign urging personal savings and voluntary spending reductions could heal "the economy". But the solidarity he was counting on was evaporating, and the rationale was transparently absurd and unequal to the crisis, which had been brought on by the oil shock and by profligate government spending and interest policy through the Vietnam era. It would not be until the advent of Paul Volcker that we would get a public servant with the courage and intellect to slay this beast, through an extremely costly campaign of squelching private investment.

So it was not Ronald Reagan who started the process of me-ism over patriotic solidarity. He was only expressing the sad consequence of a long series of failures and breaches of faith when he claimed that government is not the solution, government is the problem. So what was the alternative? The other major institutions of common action were and remain the corporation, and this era saw the valorization of capitalism as the system that works. It had the Darwinian structure and motivations that enforced effectiveness, even excellence. It was the environment that unleashed entrepreneurial freedom, then harnessed it for the common good. We know now that all this was vastly oversold, and ignored all the reasons why we have states to start with. But the pendulum had swung decisively from the public sphere to the private.

An unfortunate consequence of such a swing is that the party and ideology of privatization has little interest in fostering effective governance. So the competence of the state erodes further with time, becoming increasingly unable to do basic functions, and becoming corrupt as private interests gain relative power. Our current administration, were it not in power, would be a parody of self-serving corruption and incompetence. It is the pinacle of the Reagan revolution, and it is degrading, day by day, our ability to govern ourselves. This seems to be why these generational shifts take so long to correct. It is not only that we need to recognize the hole we have fallen into on an intellectual and scholarly level, but that enough voters (and enough extra to overcome the entrenched powers of capital in propaganda, lobbying, campaign finance, and other forms of corruption) have to have felt this in their bones to give an alternative ideology a chance to retake charge of the state and rebuild its capacity for effective action. 

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Vision Bubbles up into Perception

Visual stimuli cause progressive activation of processing stages in the brain, and also slower recurrent, rebounding activation, tied to decision-making.

Perception is our constant companion, but at the same time a deep mystery. How are simple, if massively parallel, sensory inputs assembled into cognition? What is doing the perceiving? Where is the theater of the mind? Modern neuroscience is steadily chipping away at these questions, whether one deems them "hard", unscientific, or theological. The brain is a very physical, if inaccessible, place, forming both the questions and the answers.

A recent paper made use of MEG, magnetoencephalography of electrical events in the brain, to track decision-making as a stepwise process and make some conclusions about its nature, based on neural network modeling and analogies. The researchers used a simple ambiguous decision task, presenting subjects with images of numbers and letters, with variations in between. This setup had several virtues. First is that letters and numbers are recognized, at a high level, in different regions of the brain. So even before the subjects got to the button pressing response stage, the researchers could tell that they had cognitively categorized and perceived one or the other. Second, the possibility of ambiguity put a premium on categorization, and the more ambiguous the presented image, the longer that decision would take, which would then hypothetically be reflected in what could be observed here within the brain.

The test presented. An image was given, and the subject had to judge whether it was a number, a letter, and what number/letter it was.

The researchers used MRI solely to obtain the structures of the subject's brains, on which they could map the dynamic MEG data. MEG is intrinsically much faster in time scale than, say, fMRI, allowing this work to see millisecond scale events. They segmented the observed stages of processing by some kind of statistical method into 1- position and visibility of the stimulus; 2- what  number or letter it is; 3- whether it is a number or a letter; 4- how uncertain these decisions are; 5- the motor action to press the button responding with all these subjective reports. In the author's words, "We estimated, at each time sample separately, the ability of an l2-regularized regression to predict, from all MEG sensors, the five stimulus features of interest." These steps / features naturally happen at different times, perception being necessarily a stepwise process, with the scene being processed at first without bias in a highly parallel way, before features are picked out, categorized, and brought to consciousness, in a progressively less parallel and more diffuse process.

Activation series in time. The authors categorized brain activity by the kind of processing done, based on how it was responding, and mapped these over time. First (A, bottom) comes the basic visual scene processing, followed by higher abstractions of visual perception. Finally (A, top) is activation in the motor area corresponding to pressing a response button. C and D show more detail on the timing of the various processes.

It is hard to tell how self-fulfilling these analyses are, since the researchers knew what they were looking for, binned the data to find it, and then obtained estimates for when & where these binned processes happened. But assuming that all that is valid, they came up with striking figures of cognitive progression, shown above. The initial visual processing signal is very strong and localized, which is understandable first because the early parts of the visual system (located in the rear of the brain) are well understood, and second because those early steps take in the whole scene and are massively parallelized, thus generating a great deal of activity and signal in these kinds of analysis. This processing is also linear with respect to the ambiguous nature of the image, rather than sigmoidal or categorical, which is how higher processing levels behave. That is because at this level, the processing components are just mindlessly dealing with their pixel or other micro-feature of the scene, regardless of its larger meaning. The authors state that 120 milliseconds is on average where the rough location (left or right of the screen) is decided by this low level of the visual system, for instance. By 500 milliseconds (ms), this activity has ceased, the basic visual analysis having been done, and processing having progressed to higher levels. The perception of what the letter is, (225 ms) and whether it is a letter or number (370 ms) happens roughly around the same time, and varies substantially, presumably due to the varying abiguities of what was presented.

At around 600 ms, the processing of just how uncertain the image is seems to peak, a sort of meta-process evaluating what has gone on at lower levels. And finally, the processing directing the motor event of pressing the button to specify what the subject has decided about the item identity or category (just a binary choice of left or right index finger) comes in on average also around 600 ms. Obviously, there is a great deal going on that these categorizations and bins do not capture- the MEG data, though fast, are very crude with respect to location, so only the broadest distinctions can be attempted.

The authors go on to press a further observation, which is that the higher level processing is relatively slow, and processing devoted to these separate aspects of the perceptual event takes longer and trails off slower than one might expect. Comparing all this to what is known from artificial neural networks, they conclude that it could not possibly be consistent with strictly feed-forward processing, where one level simply does its thing and communicates results up to the next level. Rather, the conclusion, in line with a great deal of other work and theorization, is that recurrent processing, making representations that are stable for some time some of these levels, is required to explain what is going on. 

This is hardly pathbreaking, but this paper is notable for the clarity with which the processing sequence, from visual stimulus to motor response, is detected and presented. While working in very broad brushstrokes regarding details of the scene and of its perception, it lays out a clear program for what comes next- filling in the details to track the byways of our thoughts as we attend consciously to a red flower. This tracking extends not only the motor event of a response, but also to whatever constitutes consciousness. This paper did not breathe the word "conscious" or "consciousness" at all, yet a video is provided of the various activations in sequence showed substantial prefrontal activity in the 450 ms range after image presentation, constituting a sixth category in their scheme that deserves a bit more attention, so to speak.

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Coronavirus M protein

Why is SARS-CoV2 such a protean virion?

Some viruses have a crystaline structure- a capsid that is absolutely uniform, dictated by the proteins that make it up in orderly arrays. The classic T4 bacteriophage is like that, and one of its features is that it stuffs its DNA contents into the capsid under extremely high pressure (20 atmospheres). That allows those contents to shoot out later with great force, into its victim- a bacterium with a strong cell wall. Coronaviruses and related influenza viruses are not like that at all. Their RNA contents are loosely collected, and the capsid assembled in cellular membranes, ending up with a variable, almost floppy, shape. The way into the next target cell is not via a physical power-injection system, but much more gently, by using one of the eukaryotic cell's several mechanisms for endocytosis to slip inside, and then veer off from the fate of being digested like food, and instead set up shop inside its own custom-made system of vesicles. 

The coronavirus surface (envelope) is composed of only a few proteins- the spike (S) protein that sticks out and attaches to, and initiates fusion with, target cells; the envelope (E) protein, which is a minor helper towards the virus's final shape; and the membrane (M) protein, which makes up most of the envelope. E is not essential, and S is not essential at all for virus formation, but M is quite essential. It gathers in huge amounts at internal membranes (called ERGIC) in rafts in preparation for virus assembly. Meanwhile the N protein has bound to special sequences on the genomic RNA, forming a sort of chromosome. The genomic RNA also has a particular tail that can bind directly to M protein. It is M that really orchestrates the whole assembly process, binding to the genomic RNA, to N, to E, and to S proteins, not to mention membrane lipids.

Schematic of the coronavirus envelope structure. The spike (S) protein in light gray sticks outwards. The nucleocapsid (N) protein in dark gray organizes the strands of genomic RNA. The envelope (E) protein in yellow occurs occasionally to impose curviture on the envelope, encouraging it to adopt a spherical shape. And the membrane (M) protein in black forms the bulk of the envelope, binding to and organizing all the other components. These authors find that occasional M proteins (red) lack inward projections. 

Incidentally, a recent paper came out about influenza M protein, which plays a very similar role, except that influenza virions are even more variable, forming into both spheres and filaments. These authors were able to generate remarkable images of the influenza M protein forming into very regular filament arrays, seen in cross-section, below. 

Filaments of M1 protein from influenza virus, see in cross-section, with a littering of half-helices lying about. The core of these filaments would be filled with infectious genomic RNA. Scale bar is 1000 nm.

So- why so unstructured? First, the viral envelope is developed from cellular membranes, and the final virus envelope still has some amount of lipids from those membranes. These tend to be quite fluid, limiting the structural regularity that can be achieved. Also, since there is no need for pressurized injection to the target, there is no need to obsess about the virus container as a totally defined, rigid body. It is apparent, from the micrographs below, that virion volumes can vary substantially, at least two-fold, suggesting that sometimes two genomes can get packed into one virion, or even more. Where is the harm in that? Given some spread by aerosols, twice the virus dose is perhaps a reasonable tradeoff for a slightly bigger and less spread-able virion container.

Is M a target for the immune system or for vaccines? No, no one seems to care about it, since it is the S protein that is most exposed on the surface and the key for attaching to and infecting new cells. Yet all parts of the virus life cycle are interesting, and it is certainly possible that small molecule drugs directed against M could be highly disruptive, to virus assembly if not to already- formed virions. Drug targets or not, these proteins play a humble structural role, knitting together virions so that they can go out into the hostile world on a puff of air and survive a few hours, enough to enter new hosts incautious enough to be gabbing in enclosed spaces.

A comparison of virions grown without S protein (top) or with S protein (bottom). Nor is N protein required for virion formation, really. It is M that organizes everything. These experiments use another coronavirus, mouse hepatitis virus (MHV).

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Eugenics, the Catholic way

Woe betide any tampering with God's nature! However, destroying it with overpopulation is OK.

The current Supreme Court battle puts a spotlight on Catholicism in law and ethics. With the impending justice, six of the nine will be Catholic. The more rightward Catholic justices are coming from a culture that has some peculiar views on itself, on key ethical issues, and on the future of the world. First is its self-righteousness. Fundamentalist Catholics like Antonin Scalia and Attorney General Barr are confident that they come to government service steeped in the most exacting and time-honored moral code- that of the church which has been in existence going on two thousand years. It is a church that has weathered millennia of political turmoil and tectonic shifts of philosophy. But does all that history make it right? Does durability imply anything other than a canny grasp of human psychology, both in keeping its parishioners in the fold, and in keeping the wheels of its authoritarian structures turning? I don't think so. Far better moral systems have been imagined and enacted, and the Church has, time after time, grudgingly taken them up, typically a century after the rest of society. Today, a Catholic woman is nominated to the Supreme Court. Maybe in a hundred years, a female cardinal? 

But what is particularly galling is the prating about the sacredness of life. William Barr has restarted federal executions, to add to all his other lying and subversions of justice, giving one a curious impression of this "culture of life". What is obviously a simple policy of patriarchal power is dressed up in gilded rhetoric of concern for "life", which, maddeningly, is swallowed as gospel by the women who are its victims. For opposition to contraception and abortion are foremost attacks on the agency and full personhood of women, who are demoted to vessels for male procreation. But the Catholic church's policy is not just patriarchy of a demeaning and sexist kind, it also constitutes a eugenic policy. Ron Turcotte, one of the great horse jockeys, born in a family of twelve children in French Catholic New Brunswick, recalled in his autobiography that the priest would make the rounds of local families and berate every woman who did not have babies in diapers. The Catholic imperative is to fill up the world with Catholics, no matter the suffering of women, families, or communities. The entire biosphere groans under vast overpopulation. And what is the answer of the Catholic church? More Catholics, more oppression, more mental straightjackets. Care for creation apparently does not extend to continence on the part of men, basic personal rights or autonomy on the part of women, or to creation in general.

Just another day at the Supreme Court.

So when I hear "distinguished" lawyers, scholars and ethicists from Catholic institutions pontificate about the evils of genetic engineering, stem cell research, or use of embryos in research, (not to mention abortion or assisted suicide, among many other topics) I can not take them seriously as intellectuals- as anything other than mouthpieces of an antiquated system of oppressive, and now catastrophic, archetypes of political and social power. It is one thing to be a scholar of an artistic tradition full of glorious human expression and yearning quests for deeper connection with whatever power animates the world. But with the loss of humanism, then Protestantism, Catholicism retreated into an intellectual fortress of defense, nostalgia, and counter-reformation. The Federalist societies, the constitutional textualists, the Opus Dei fundamentalists... this ecosystem that has funded and nurtured a conservative assault on US legal institutions, apparently heavily Catholic, all are backward time machines fixated on dead controversies and traditional, frankly eugenic, policies of world domination. 

Nominee Barrett's textualism, following Scalia, seems to endanger the last century or two of constitutional interpretation. Whatever is not explicitly enumerated in the text is not, by this view, in the federal government's power. This could include women, (other than the 19th amendment; notably, the word "he" is used repetitively to refer to the president, representatives and other officers), federal regulatory authority in countless areas such as labor, antitrust, and finance, and the very meanings of concepts like cruel and unusual punishment, militias, privacy, due process, "needful rules", and "general welfare". The constitution and statutes are frequently vague, precisely so that society can construct its meanings according to the spirit of the document, not a cramped view of its letter, or a psychoanalytic plumbing of its mental conditions of origination.

Nor is Catholicism alone in this backwardness and revanchism. Islam shares its authoritarian, righteous, patriarchal, misogynistic, domineering mentality, even while lacking a pope. It goes the Catholics one better by approving of polygamy, another eugenic gambit. Consequently, Islam has even higher birth rates than Catholicism, immiserating its populations, stoking misplaced resentment, and imperiling the biosphere. However, Muslims in the US are not at this time constructing legal pipelines into US federal judgeships or dominating the Supreme Court, so their similarities in this regard are of global, but not federal, concern.

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