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Introduction to micro-RNA
Why It Got Lost in the Shuffle for so long
 
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The Fault with the Methods for Isolating and Observing RNA's:

It seemed quite a surprise in January 2002 that cells were reported to be filled with various RNA's that are not translated (ref). Due to stereotyped thinking for the past two or three decades, so much attention has been focused on mRNA, which is translated, that it seemed to be forgotten that MOST of the RNA in normal cells is not translated. In an E.coli cell, for example, 95% of the RNA is not translated! Of that 80% is ribosomal RNA (rRNA), and 15% is amino-acid transfer RNA (tRNA). At best mRNA never makes up more than a few percent of the total RNA in a cell. However, even those statistics are suspect because they were derived using methods that would have overlooked the μ-RNA's - indeed, discarding it as "junk" or unwanted breakdown products. Let's see how this came about: let us look at the early methods which established the stereotyped mindset.

PHENOL EXTRACTION OF NUCLEIC ACIDS. Nucleic acids initially were isolated using the "phenol method" invented by Gierer and Schramm (ref), who used their method for that milestone work showing that the RNA inside of TMV (tobacco mossaic virus) was the genetic material. They were primarily interested in designing a method that cleansed nucleic acid of all protein, because in those days there was yet a great deal of incredulity that the simple nucleic acid "code" was complex enough to be the code of life. Proteins were far more complex and many stuck to the idea that genes were really made of protein. So you can see why it was so important that all traces of protein be removed from their TMV preparations, which were shown to contain RNA. The phenol method denatures and removes the protein from the aqueous phase, leaving all sorts of salts, sugars, amino acids, other small molecules and nucleic acids of all types and sizes in solution in the water phase. Finally, they used alcohol to precipitate the macromolecules from that mix. And there comes the problem: the μ-RNA's were too small to readily precipitate, and would not form floc to be centrifuged out, and, unlike DNA, could not be drawn out by spindling on a stirring rod.

Subsequent additional methods such as the amyl alcohol or isobutanol methods were even less able to precipitate out μ-RNA's as they were designed mostly for isolating huge DNA molecules and leaving more of the contaminating RNA behind.

Nobel laureate Hershey and his team sought methods for purifying RNA of any contaminating DNA. Previously this was tricky because nucleases were employed to destroy the "other" type of nucleic acid. If you wanted DNA, treat with RNase; and if you wanted RNA, then treat with DNase. BUT there was a problem: While RNase can be easily purged of DNase activity by bringing a solution of RNase to a boil, and then cooling and allowing the small RNase molecules to renature, it was a problem getting DNase "clean" of RNase activity. Usually RNase inhibitors were added to eliminate that unwanted activity from DNase digestions of RNA preparations. In any event, both types of nucleic acid preparations were contaminated with the breakdown products of the other sort. Again, alcohol or trichloroacetic acid precipitations were the preferred methods to flush away the unwanted small molecular debris.

"MAK" COLUMNS. Hershey's group then tried another method and invented a chromatographic column they cleverly named the "Hershey Column" (while other lighthearted labs called it the "chocolate column"), which finally came to be known as a "MAK column" because it was made of methylated albumen on kieselguhr (diatomaceous earth, or filter aid). This worked wonderfully for their purposes: DNA stuck irreversibly, and the various sized RNA's could be eluted with higher and higher salt concentrations. Giving curves like those shown to the right. Nevertheless, notice the very high initial peak. Yup! Disregarded small "junk" RNA's! Detailed explanation: This is a composite graph combining RNA data from both prokaryotes and eukaryotes. Both realms of life present the same profiles for their tRNA's - a double lobed peak that elutes at low concentrations of salt. However, as the salt concentration steadily increases, differences appear in the rRNA's of the two realms of life. While the column separates the two prokaryotic rRNA's, it does not with the two eukaryotic rRNA's, which come out as one peak. Another method was needed to separate those, and that is shown next. But before turning to that, it must be asked where the mRNA is on this profile of "all" RNA's? They were found to reside in a very broad (heterogeneous) band between the tRNA's peak, and the rRNA's peak(s) - with most a bit closer to the rRNA peak(s).

SEDIMENTATION CENTRIFUGATION. An alternative way to look at RNA's and not DNA's were by radioisotopically labelling the RNA's with carbon-14 tagged uridine, and then using sedimentation centrifugation, which sorted RNA's by size - the larger the molecule the faster it settled based on Svedberg's principles that rate of settling was proportional to the square of the molecular weight. It is important to note the difference between sedimentation and density gradient centrifugation techniques. The former is a dynamic system, which if the centrifuge is forgotten, all of the material will eventually get to the bottom of the tube. Density gradient centrifugation reaches an equilibrium so that each type of particle eventually comes to rest (or float) at its own level of specific gravity (iso-pycnos [density]). In the latter case, the centrifugation is only used to speed the molecules in finding their corresponding levels in the gradient.

Detailed explanation: This requires an ultracentrifuge that runs about 20 krpm. Thin plastic tubes are used. (A) Into a tube, a gradient of sucrose is loaded such that at the meniscus the concentration is 5% and it increases in concentration linearly to 20% at the very bottom of the tube. In this way, not only the density but also the viscosity of the solution increases with depth in such a way that vertical mixing in minimized and the increasing G-forces are continuously counteracted as the molecules sink to greater depths (radius of rotation has increased). (B) Very carefully, a sample of RNA of less than 0.5 ml is floated on top of the gradient. (C) Then to prevent the sides of the tube from rolling inwards under the high G-forces, the tube is overlayered with oil, which, of course, floats atop the aqueous phases below. Then the tube - along with two others - is spun at 20krpm in a vacuum (to minimize drag and frictional heating) for approximately 18 hours. (D) Were you able to look at the tube in ultraviolet light (approx 260nm), you would see that the RNA's have moved out of the top aqueous sample phase, and moved into the sucrose gradient. The larger the molecules, the further they have sunk (contrary to Galileo, but that's another story about why the first men on the moon dropped the hammer and the feather - no atmospheric drag!) (E) Finally the thin plastic tube is impaled on a hypodermic needle and the exiting drops are caught in a series of test tubes - perhaps 15 drops per tube (and as the historical lab lore goes - the telephone always rang with no one else to answer it; distraction and drops miscounted...!) The tubes were then assayed for either or both radioactivity and/or UV absorption at 260nm resulting in the next next graph.

When the final tube was punctured in the bottom and samples were dripped out, there were found radioactivity profiles like those shown to the right. Again the small, radioactive "junk" is seen at the top where very small molecules would be expected. It was disregarded as either breakdown products or only partially made RNA's. Detailed explanation: The results follow fairly closely the appearance of Hershey's MAK column chromatography with one major exception - sucrose gradients do separate the two species of eukaryotic rRNA. (It should be mentioned here that rRNA's can be rather easily purified directly from isolated ribosomes: merely use a phenol extraction of the ribosomes. The r-proteins are gotten rid of and the only macromolecules left are the 3 types of rRNA's.)

MOLECULAR SIEVES. On final method often employed in the "golden ages" for separating macromolecules from smaller molecules was one using molecular sieves such as one or another of the various Sephadexes (explanation and analogy). Unlike usual sieves consisting of screens which hold back large particles, molecular sieves do just the opposite by allowing the macromolecules to speed through leaving the small stuff in the column material. So once again, we see that things smaller than tRNA's escape notice to be discarded.

As some of these methods and their derivatives are still used after 40 years, it is no wonder that μ-RNA's escaped attention. They were regarded as breakdown "junk" and discarded.

NEXT PAGE

Gierer, A. and G. Schramm. 1956. Infectivity of Ribonucleic Acid from Tobacco Mosaic Virus. Nature 177: 702-703. Return to previous reading place.

More References

 
1. A review on chromosome organization
    
2. S. Brenner et al., "Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome," Nature, 366:265-8. 1993.
    
3. R. Hinegardner, "Evolution of cellular DNA content in teleost fishes," American Naturalist, 102:517-23. 1968.
    
4. G. Elgar et al., "Generation and analysis of 25 Mb of genomic DNA from the pufferfish Fugu rubripes by sequence scanning," Genome Research, 9:960-71. 1999.
    
5. M Hattori, T.D. Taylor, "Part three in the book of genes," Nature, 414:854-5, 2001.
    
6. P. Deloukas et al., "The DNA sequence and comparative analysis of human chromosome 20," Nature, 414:865-71, 2001.
    
7. Small RNA's in the news, July 2002.

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