The Futile Cycle

I have now seen molecular biology applied to Real Life.

I live in a graduate student dorm (for now), and just yesterday, the bathroom I share with other students on my floor was broken. Mainly, when water went down the sink, black, dirty water would bubble up from the shower floor drain. Gross. Luckily, flushing the toilet didn’t cause any similar problems. But by that serendipitous sewage problem, I learned that the drain of the sink fed directly into the same pipe system that the shower drain did, while the toilet drained into a separate pipe system (or it tied in further downstream of the drainage problem).

In summary, I found one way that the system broke (a mutation) that led to interesting behaviors (a phenotype), and by characterizing these behaviors, I gained a better understanding of how that system worked (a mechanism).

Molecular biology! In the real world!

Small interfering RNAs (siRNAs) have been going through an interesting revival period as of late. In the past two months, there have been at least six high profile papers in Nature and Science on siRNAs. True, we’ve already seen siRNAs long ago; in fact, the 2006 Nobel Prize in Medicine went to Andrew Fire and Craig Mello for their work in discovering siRNAs in animals. The thing is, after Fire and Mello’s initial work, many people thought that RNA interference (i.e. the process by which siRNAs inhibit genes) was an evolved anti-viral mechanism that just happened to have a very general, highly evolutionary conserved, and extraordinarily useful gene regulatory “side effect” that researchers could use for manipulating and studying biology. It sounds too good to be true.

It was.

Such a useful gene regulatory mechanism just couldn’t sit around as a “side effect” for very long. Evolution, by random mutations, is constantly exploring many different ways of regulating gene expression, so it stands to reason that animals might have evolved ways to regulate genes with siRNAs. The only problem is that no one has found any endogenous siRNAs — until recently, of course.

The five papers in Nature (Watanabe et al., Murchison et al., Czech et al., Okamura et al., and Kawamura et al.), and one paper in Science (Ghildiyal et al.) all describe how they separately discovered these native small RNAs that function to regulate gene expression. This is no anti-viral mechanism or some weird gonad-specific thing; this is genuine gene regulation! The papers span both fruit flies and mice.

It’s surprising, really, that these discoveries didn’t come about sooner, but I suppose the technical innovations here and there are what led to these discoveries. One of the biggest factors that probably contributed to the new wave of siRNA discoveries is the coming of next-generation sequencing technology, which allows for sequencing of lots and lots of really short pieces of DNA and RNA, perfect for looking at small RNAs.

The discovery two years ago of endogenous siRNAs in nematodes using this technology also helped spur this new wave. No one was sure whether these endogenous siRNAs were specific to worms, or whether other animals had these traits. Worms had one trait they shared with plants and fungi that other animals don’t: an RNA-dependent RNA-polymerase (RdRP), which amplifies siRNA signals. Worms, plants, and fungi had endogenous siRNAs, but because of the differences in the players, it was certainly possible that they had something that mammals and fruit flies didn’t. The next step, of course, was obvious enough that it led to this new flood of papers, all using pretty straightforward bioinformatics, biochemistry, and sequencing to scour fly and mouse genomes for siRNAs.

For a while, the hotness factor of RNA was starting to wear off in the field, but it seems like interest might come back in a new wave. I’m game for another revival.

Yet another article from Nature today on RNA therapeutics, this time on using RNAi to stop angiogenesis in the eye to prevent blindness. Some people have seen that the VEGFR receptor can be targeting for knockdown by RNA interference using short 21-nucleotide siRNAs. Apparently, no one bothered to do the control here.

The authors of this paper did the control, in which one uses a “scrambled” or off-target siRNA to show that the effect of the silencing is sequence-specific. Except, in this case, the effect wasn’t sequence-specific. In fact, any old RNA would work, as long as it was longer than 21 nucleotides.

This might ring some bells about innate immunity. One of the early problems with RNAi in humans was that long double-stranded RNAs, which can be chopped up in cells to form the siRNAs, cause human cells to become inflamed. Specifically, the RNAs activated some Toll-like receptors, leading to a mounting of the innate immune response. This immune response was originally evolved to combat RNA viruses, which often have double-stranded RNA genomes or go through a double-stranded RNA intermediate during infection. This problem was later solved by using pre-made short RNAs, which don’t really induce the immune system response.

In this paper, it seems the authors have found this effect at play again. Many of the RNAs they tried activate the immune response, which in turn causes the cell to suppress angiogenesis!

Yesterday in Nature was a really exciting paper on miRNA-targeting therapeutics: Locked-Nucleic Acid-based knockdown of miRNAs in vivo!

microRNAs (miRNAs) are really tiny regulatory RNAs (about 22 nucleotides long); efficient, specific hybridization would normally require something much longer. Recently, though, the use of “locked nucleic acids” has become more popular. These are RNA analogues that have an extra bridge in the ribose sugar, making oligos of them rigid. Entropically, this greatly enhances binding of the LNAs to the RNAs, which means that one can use them for things like in situ hybridization much more easily and specifically! Not only that, but the use of LNAs instead of normal RNAs means that the half-lives of the oligos become much longer, similar to what one would see with morpholinos.

The authors injected LNAs into monkeys in order to target miR-122, which regulates cholesterol metabolism (among other things). They managed to effectively silence the miR-122 and they showed a drop in cholesterol levels!

Very exciting stuff!

This is really quite odd. In today’s Science
bacteria have been found to nucleate many ice particles in the atmosphere, including snowflakes. In other words, bacteria are raining and snowing from the sky!

There are two interesting new papers out in the newest issue of Molecular Cell, and one from Science a week back that’s online early:
An Important Role for the Multienzyme aminoacyl-tRNA Synthetase Complex in Mammalian Translation and Cell Growth: Seems that the two different forms of Arginine tRNA synthetase have different roles in the cell, one to make tRNAs for synthesizing proteins, and to make tRNAs for degrading them via the ubiquitin pathway!

Human Alu RNA is a Modular Transacting Repressor of mRNA Transcription During Heat Shock:
Non-coding RNAs have been shown to have transcriptional regulatory properties, but this particular paper discusses one that looks a lot like a protein, in the sense that it seems to have modular “domains.” Pretty neat work!

Selective Blockade of MicroRNA Processing by Lin-28: The main discovery of the paper is, of course, nicely summarized in the title. The idea is that Lin-28 prevents the biogenesis of let-7, one very well-studied (though not well-understood) family of microRNAs, by binding to the RNA and preventing Drosha and Dicer from gaining access to it.

Day to day, I work a lot with clear, colorless drops of liquid, shuttling them back and forth between small plastic vials. I also work with cells, but they just look like cloudy suspensions in strange-smelling liquids.

But every time I look at cells under a microscope, I can’t help but gaze in wonderment. Cells! Under a microscope!

Even better are images and videos online from the American Society for Cell Biology, which I found via Bitesize Bio. Make sure to check out the videos, some of which are absolutely spectacular and inspiring. Some of my favorites include watching rat heart cells beating in a Petri dish, seeing fish cells zooming across slides (especially check out how fragments of cells without a nucleus can still move around! Movement is thus, at least in the short term, independent of transcription), the movement of mitochondria within a cell, seeing chromosomes divide in real time using just light microscopy, and watching Drosophila embryo syncytial division (where all of the nuclei divide simultaneously, without cytokinesis).

Biology is an absolute wonder, and I hope you find these videos as inspiring as I do.

Lately, genome-wide association studies are popping up everywhere. Just scanning through the latest Nature Genetics, almost half of the articles are such linkage studies. The studies represent one of the greatest convergences of population genetics, fundamental molecular biology, the human genome project, the HapMap project, disease biology, and microarray technology.

Leonid Kruglyak has a great review article out in Nature Reviews Genetics on the history and development of such genome-wide studies.

I think these kinds of studies will eventually have the potential to revolutionize medical diagnostics and drug therapy, since it’ll become easier and easier to figure out risk factors for disease and tailor drug therapies to the specific risk categories a patient falls into. I’m really excited to see how this field progresses, especially when newer technologies arise for rapid sequencing of genomes!

Pure Pedantry links to an awesome paper on visualizing the cell cycle in mice using fluorescent markers. They can even look at the cell cycle state of cells in histological sectionsof mice! Check out the blog post, because Jake Young highlights the coolest picture and the coolest video in the paper; the movie at Cell and ScienceDirect seem to be down, so you can see the video at Pure Pedantry or at Google Video.

One of my biggest fans got me a copy of The Eighth Day of Creation, which I’d been wanting to read for some time now. In the first chapter, check out this old-timey maxiprep (i.e. large-scale DNA isolation and purification) protocol, courtesy of Avery, MacLeod, and McCarty from their historic 1944 paper demonstrating that DNA was the carrier of genetic information in cells:

To get [DNA], they grew virulent Type III pneumococci at blood heat in twenty-gallon vats of broth made from beef hearts, spun out the bacilli in an iced centrifuge, suspended them in brine, and brought the “thick, creamy suspension of cells” quickly to a temperature hot enough to kill the cells and to inactivate “the intracellular enzyme known to destroy the transforming principle” [DNase]… They then washed the cooked pneumococci in three changes of brine to remove capsular sugar as well as whatever protein would come away, extracted the bacteria by shaking them for an hour in a solution of bile salt to break the cell walls (and then threw away the cell residue), and reprecipitated the extract with pure grain alcohol.

“The precipitate forms a fibrous mass which floats to the surface of the alcohol and can be removed directly by lifting it out with a spatula,” the paper said. This was now washed several times with chloroform to remove protein, and suspended yet again. A digestive enzyme was put in to eat away any remaining capsular sugar. Removal of protein was repeated, “until no further film of protein-chloroform gel is visible at the interface.” Pure grain alcohol was added again, “dropwise to the solution with constant stirring.” At a concentration where the alcohol nearly equalled the extract, “the active material separates out in the form of fibrous strands that wind themselves around the stirring rod. This precipitate is removed on the rod and washed….The yield of fibrous material obtained by this method varies from ten to twenty-five milligrams per seventy-five liters of culture.”

Wow, and I thought maxipreps take too much time now! Compare the above to a more modern DNA isolation protocol, courtesy of Black Knight.