Putting the polyadenylation complex together

One of the reasons for the slow pace of entries here has been the intrusion of, um, life into my life.  (Yeah, figure that one out.)  Earlier this summer, a paper from my lab was published in BMC Cell Biology.  Seeing as it’s Open Access, and since it has a bit of relevance to a theme introduced in this essay, I thought I would point it out here.

The story in a nutshell – one of the subunits of the polyadenylation complex is the so-called CPSF30 protein, or its yeast relative Yth1.  (Yth1 looms large as one of the few subunits in the Giardia complex.)  What Drs. Suryadevara Rao and Randy Dinkins did was study the places within the cell where CPSF30 goes, and what happens when one co-expresses this protein with other polyadenylation complex subunits.  They did this by attaching the various proteins to fluorescent proteins and following the fusion proteins using microscopic techniques.

The results corroborated other studies that detailed interactions between various of these proteins.  However, a rudimentary deletion analysis showed that these interactions by and large involve parts of CPSF30 that are not found in the mammalian or yeast proteins.  Since the CPSF30 interacts with the other proteins of interest in this study (the 160, 100, and 73 kD subunits of the cleavage and polyadenylation specificity factor, or CPSF) in other eukaryotes, it stands to reason that the interactions themselves must have evolved independently.  This in turn suggests a somewhat different trajectory in the evolution of the complex in different eukaryotic lineages.  It also raises the possibility that the different complexes may process and polyadenylate RNAs in subtly different ways.

Some pretty pictures and a link to a fascinating movie may be found beneath the fold.  Enjoy.

Meanwhile, back in the RNA World …

I’ve been traveling a lot lately. I don’t have time for some, um, wordy essays, but time hasn’t stood still. So I thought I would point out some interesting stuff that has appeared recently. These have an RNA World theme.

If there is a message I wish to send, it is that the RNA World is, as always, a thriving and exciting place for a scientist to be. Enjoy. (The last two entries are going to generate lots of buzz in the blogosphere.)

On the utility of evolution in experimental biology and medicine

A recurring theme amongst ID antievolutionists holds that evolution really doesn’t contribute useful directions or concepts in the realm of biology or medicine. Philip Skell regurgitates the theme in a recent commentary in Forbes magazine:

“Examining the major advances in biological knowledge, one fails to find any real connection between biological history and the experimental designs that have produced today’s cornucopia of knowledge of how the great variety of living organisms perform their functions. It is our knowledge of how these organisms actually operate, not speculations about how they may have arisen millions of years ago, that is essential to doctors, veterinarians, farmers and other practitioners of biological science.”

And later:

“The essence of the theory of evolution is the hypothesis that historical diversity is the consequence of natural selection acting on variations. Regardless of the verity it holds for explaining biohistory, it offers no help to the experimenter–who is concerned, for example, with the goal of finding or synthesizing a new antibiotic, or how it can disable a disease-producing organism, what dosages are required and which individuals will not tolerate it. Studying biohistory is, at best, an entertaining distraction from the goals of a working biologist.”

The blogosphere (and probably print media) are replete with summaries and specific cases that show Skell’s assertions to be a crock. This essay summarizes one such example. I have chosen this one because it refutes, specifically, the claim that an understanding of the evolutionary history of an organism “offers no help to the experimenter–who is concerned, for example, with the goal of finding or synthesizing a new antibiotic, or how it can disable a disease-producing organism”. It also ties Skell’s uninformed comments in with another subject that causes ID antievolutionists much consternation – the origins and evolution of organelles.

Behe and the limits of evolution

Intelligent Design proponent Michael Behe has recently taken Ken Miller to task for the latters rough handling of another ID proponent’s handling of some concepts in evolution.  I don’t intend to add to the back and forth between the two (or three?) of them here.  Rather, I thought I would use one of Behe’s closing remarks as an excuse to repost a (slightly-modified) Panda’s Thumb essay that pertains to one of Behe’s newer calling cards – the so-called “Edge of Evolution”.

In the last paragraph of his response to Miller, Behe says:

“It’s pertinent to remember here the central point of The Edge of Evolution. We now have data in hand that show what Darwinian processes can accomplish, and it ain’t much.”

Actually, as the following essay clearly shows, Darwinian processes can do much more than Behe suggests.  Enjoy.

Where does all that DNA go?

In this recent essay, I discussed studies that showed a surprisingly high rate of movement of DNA from organelles to the nuclear genome.  Curious and questioning readers should have wondered about this, as one implication is that nuclear genomes should become huge mosaics of organelle DNA in a relatively short evolutionary time.  Of course, this is not the case – organelle DNA may be found in nuclear genomes, but it makes up a tiny fraction of these genomes.

If you had read my essay and wondered along these lines, you may pat yourself on the back.  For this paradox is something that has puzzled others.  A recent paper in PLoS Genetics helps to resolve things.  Briefly, Anna Sheppard and Jeremy Timmis followed up on the earlier studies , asking what happens to the organelle genes after they wander into the nucleus.  These authors found a high frequency with which the organelle DNA (at least the marker gene that was followed in these studies) is altered or deleted.  As the authors discuss (the abstract follows beneath the fold), this suggests that the overall picture is a dynamic one – DNA can move into the nucleus at a high rate, but it is also removed relatively rapidly.  The result is a sort of steady state, one that affords the creation of new nuclear genes without the burden of vast amounts of organelle DNA.

Axe (2004) and the evolution of enzyme function

[Preface – the subject of protein evolution pops up on a regular basis in ID circles.  Recently, William Dembski mentioned the study alluded to in the title of this essay as an improved argument/piece of evidence for intelligent design.  Specifically, Dembski said:

“(2) The challenge for determining whether a biological structure exhibits CSI is to find one that’s simple enough on which the probability calculation can be convincingly performed but complex enough so that it does indeed exhibit CSI. The example in NFL ch. 5 doesn’t fit the bill. The example from Doug Axe in ch. 7 of THE DESIGN OF LIFE (www.thedesignoflife.net) is much stronger.”

“The example from Doug Axe in ch. 7 of THE DESIGN OF LIFE” would appear to be Axe’s 2004 paper in the Journal of Molecular Biology, the subject of my first ever essay on The Panda’s Thumb.  Since I have been a bit remiss in re-posting older essays here, I thought I would use this excuse to put this here.  It’s “published” without change, so as to maintain some sort of continuity.  As always, enjoy.]

Douglas Axe recently (well, sort of) published an article in the Journal of Molecular Biology entitled “Estimating the Prevalence of Protein Sequences Adopting Functional Enzyme Folds” (Axe, J Mol Biol 341, 1295-1315, 2004). In his discussion of the experimental observations, Dr. Axe mentions some numbers that are likely to generate much discussion amongst Intelligent Design advocates and critics. For example, Stephen Meyer (2004) cites Axe at a key point in the argument in his recent article advocating Intelligent Design, “The Origin of Biological Information and the Higher Taxonomic Categories,” much discussed in previous Panda’s Thumb threads (here).

“Axe (2004) has performed site directed mutagenesis experiments on a 150-residue protein-folding domain within a B-lactamase enzyme. His experimental method improves upon earlier mutagenesis techniques and corrects for several sources of possible estimation error inherent in them. On the basis of these experiments, Axe has estimated the ratio of (a) proteins of typical size (150 residues) that perform a specified function via any folded structure to (b) the whole set of possible amino acids sequences of that size. Based on his experiments, Axe has estimated his ratio to be 1 to 10^77. Thus, the probability of finding a functional protein among the possible amino acid sequences corresponding to a 150-residue protein is similarly 1 in 10^77.”

More recently, Dembski cited Axe in his Expert Witness Report for the Dover trial (see this).

“Recent research by Douglas Axe (see Appendix 3) provides such evidence in the form of a rigorous experimental assessment of the rarity of function-bearing protein sequences. By addressing this problem at the level of single protein molecules, this work provides an empirical basis for deeming functional proteins and systems of functional proteins to be unequivocally beyond Darwinian explanation.”

Given that this subject is often raised by ID proponents (such as this), and that the Biologic Institute (where Axe works) has made some news accounts, it seems appropriate to review Axe’s work. The purpose of this PT blog entry is to try and lay out the study cited above (Axe DD, J Mol Biol 341, 1295-1315, 2004) in a form that is accessible to most interested parties, and to discuss a larger context into which this work might be placed. Needless to say, the grand pronouncements being made by the ID camp are not warranted.

RNA and kitchen tools – Slicers, Dicers, and CRISPRs

RNA-based regulation is all the rage in biology today.  The more familiar mechanisms involve small RNAs such as microRNAs and silencing-associated RNAs.  The biogenesis and functioning of these RNAs involves enzymes and complexes that have been termed, among other things, Dicers and Slicers.  These subcellular kitchen utensils work by processing either the small RNA precursor or the base-paired target RNA.  This mode of regulation is most often associated with eukaryotes, and indeed homologous enzymes and mechanisms are not found in prokaryotes. However, systems with remarkable functional similarity may occur in bacteria.  A recent review by Sorek et al. brings one such example into focus.

One curious feature of bacterial genome is the occurrence of arrays of direct repeats in which the repeated units are separated by so-called spacers of unique sequence unrelated to the repeat units.  The sizes of the repeat units vary from bacteria to bacteria, ranging from between 24 to 47 bp.  Likewise, the spacer sizes vary from 26-72 bp.  These arrays are flanked by an apparent leader sequence, and yet again by arrays of protein-coding (CAS) genes, the number and composition of which vary considerably from bacteria to bacteria.  The general arrangement is shown in the following figure, which is part a of Figure 1 from Sorek et al. (shown beneath the fold):

Is macroevolution impossible to study (Part 2)?

The plant kingdom is many things – the basis of agriculture and civilization, a natural laboratory with a stupefying capability in organic synthesis, a source of untold numbers of pharmaceuticals, antimicrobials, herbals, and other chemical playthings, a fascinating range of biological form and function, and an eminently accessible subject for studies of evolution. Along the lines of the last two bullets, one of the more interesting aspects of plants is the range of growth habits that may be adopted. Among these are two sets of contrasting characteristics – annual or perennial, and herbaceous or woody. Differences in these characteristics are among the bases for classification of plant species. For this reason, but also because accompanying morphological differences can be quite considerable, evolutionary changes that involve transitioning between these states are macroevolutionary. Thus, it stands to reason that studying the means by these characteristics evolve amounts to experimental analysis of macroevolution, and understanding the underlying mechanisms constitutes an explanation of macroevolutionary processes.

It is in this light that a recent report deserves some attention. This report, by Melzer et al., describes studies of the functioning of two regulators of flowering in the herbaceous annual Arabidopsis thaliana. These proteins, called SOC1 and FUL, had been known for some time to be involved in the regulation of flowering. Melzer et al. constructed double mutants deficient in the expression of these two proteins, with the intent of understanding the physiological significance of interactions between these two proteins, associations discovered using the so-called yeast two-hybrid assay. Amazingly, soc1 ful double mutants were dramatically different – they had a more woody growth habit, and they behaved like perennials when it comes to reproduction. The abstract from the paper follows this paragraph. The bottom line that is in keeping with the title of the essay – not only can this particular macroevolutionary process be studied experimentally, it can be understood and the corresponding macroevolutionary process recapitulated in a controlled setting.

The abstract:

Plants have evolved annual and perennial life forms as alternative strategies to adapt reproduction and survival to environmental constraints. In isolated situations, such as islands, woody perennials have evolved repeatedly from annual ancestors1. Although the molecular basis of the rapid evolution of insular woodiness is unknown, the molecular difference between perennials and annuals might be rather small, and a change between these life strategies might not require major genetic innovations2, 3. Developmental regulators can strongly affect evolutionary variation4 and genes involved in meristem transitions are good candidates for a switch in growth habit. We found that the MADS box proteins SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) not only control flowering time, but also affect determinacy of all meristems. In addition, downregulation of both proteins established phenotypes common to the lifestyle of perennial plants, suggesting their involvement in the prevention of secondary growth and longevity in annual life forms.

The citation:

Melzer S, Lens F, Gennen J, Vanneste S, Rohde A, Beeckman T. 2008. Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana. Nature Genetics, published online: 9 November 2008 | doi:10.1038/ng.253

Transplastomics – a convergence of biotechnology and evolution

One of the going concerns in plant biotechnology is the matter of containment of transgenes in the field.  This concern arises from the inescapable fact that genetically-modified crop plants may, depending on the specific species involved, “disseminate” transgenes via hybridization with nearby plants (of the same species or closely-related ones).  A number of strategies have been devised to reduce or eliminate this possibility.  Among these is the approach of placing transgenes in the chloroplast genome of a recipient crop plant.  The rationale behind this approach lies in the fact that the chloroplast genome is inherited in a maternal fashion (much as are mitochondrial genomes in animals).  Consequently, pollen shed by a transplastomic plant (the jargon shorthand term for the plant that has one or more transgenes resident in the plastid genome, as opposed to the nuclear genome) should not carry or transmit the transgene, since transmission is through the female gamete.

Expressing foreign genes in the chloroplast comes with some other advantages.  Since the chloroplast is a prokaryotic genetic system, it is not “encumbered” by the presence of an elaborate and hard-to-control gene silencing system, one that affects nuclear-sited transgenes in a haphazard fashion.  This means that expression of chloroplast-situated transgenes is more consistent (and often attains higher levels) that that of similar nuclear transgenes.  The chloroplast is as well the location for some very highly-expressed proteins (plant physiology students learn early on that the chloroplast enzyme rubisco aka ribulose-1,5-bisphosphate carboxylase is the most abundant protein on earth), which means that it is feasible to attain higher protein levels in such systems than from nuclear transgenes.  (Of course, there are controls on mRNA and protein accumulation in the chloroplast, so that it is necessary to test and manipulate the specific transgene and its protein product to achieve the desired results.)

These considerations aside, the possibility of transmission of chloroplast-sited transgenes remains something of an open issue.  One matter should be familiar to readers who follow the field of human ancestry; work in this field has been complicated by the observation that mitochondrial genomes, typically assumed to be maternally-transmitted, may on occasion be inherited through the paternal gamete.  A similar concern applies to those plant species that are assumed to transmit chloroplast genomes maternally; for example, recent studies (5, 8 ) show that paternal inheritance of chloroplast-localized transgenes does occur.

An interesting take on poly(A) signals

One interesting facet of RNA biology is the matter of the occurrence and function of structural RNA units within RNAs inside of living cells.  Excellent examples of these are the many so-called riboswitches, motifs that bind metabolites and alter the functionalities of RNAs in which they reside.  As more and more examples of RNAs with catalytic activities become known, questions naturally arise as to whether such activities might impact RNA function in vivo.  A recent study poses just such a question for what is among the simplest of known catalytic RNAs, namely the manganese-dependent ribozyme.  This enzyme consists of little more that a GAAA-UUU complex.  These two motifs, that need not be adjacent in the RNA, are expected to occur at a high frequency in natural RNAs.  A recent report indeed finds that RNAs that possess such motifs indeed may be Mn-dependent ribozymes.  The physiological signifiance of this finding is, in my opinion, a bit of an open issue.  But the possibilities are fascinating.  The last sentence of the abstract (that follows) is especially provocative.