Messenger RNA is the link between the genome and the cell cytoplasm. For this reason alone it is of central importance in the study of gene action. This study has been greatly facilitated by the methods which are now available for the isolation and purification of mRNA’s (and other RNA’s as well). Purified mRNA is, of course, a prerequisite for experiments for the production of complementary DNA by the use of reverse transcriptase, and for experiments involving DNA/RNA hybridization.
Isolation of RNA involves firstly the disruption of the tissue and then separation of RNA from its association with proteins. During the isolation special precautions must be taken to inhibit the degradative action of nucleases. A typical method for isolating RNA is to disrupt tissues in a mixture of buffer, phenol, and an anionic detergent such as sodium dodecyl sulfate (SDS).
Inhibitors of RNase activity, such as diethyl pyrocarbonate or heparin, may be included in the buffer. The SDS releases RNA from protein complexes and inhibits RNase activity. Phenol denatures protein, which either becomes insoluble or goes into the phenol layer.
When chloroform (with iso-amyl alcohol and 8 OH quinoline) are added to the phenol-SDS mixture, the RNA, including poly-adenylated mRNA (poly A+ mRNA), is found in the aqueous phase, from which it can be precipitated with ethanol. Any contaminating DNA or protein may be removed from the RNA by treatment with DNase and proteinases.
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Poly A+ mRNA can be isolated by passing the RNA extract through an affinity column containing stretches of complementary base, e.g., oligodeoxythymidine-bound cellulose. Because of the complementarity of adenine and thymidine, the poly A+ mRNA binds to the column at high salt concentrations (0.5 M KCl, 0.01 M tris HCl) and is eluted at low salt concentrations (0.01 M tris HCl).
Nuclei and polyribosomes often are first isolated from disrupted tissues by density gradient centrifugation, so that fractions of nuclear RNA and polyribosomal RNA can be prepared.
The best chances of isolating and studying a specific mRNA are to be sought in cells and tissues that synthesize only a limited number of proteins or possibly only one kind of protein. Fortunately, some progress in this direction has already been achieved.
Messenger RNA has been isolated and purified which directs the synthesis of the silk fibroin, produced by silk glands of pupating caterpillars of the silkworm, Bombyx mori. Advantage was taken of two favorable circumstances.
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Firstly, in the posterior part of the salivary gland of the pupating caterpillar, the silk fibroin is practically the only protein produced. Secondly, silk fibroin is a very peculiar protein; most of it consists of repeating units made up predominantly of only a few amino acids. Glycine comprises 45 per cent of all residues, and it alternates predominantly with alanine and serine.
If the RNA codons for these three amino acids are considered (GGX for glycine, GCY for alanine, and UCZ or AGUC for serine, where X, Y, and Z are third bases in the triplets which do not change the meaning of the code), it can be calculated that the messenger RNA for a protein of this composition should have an unusually high proportion of guanine and cytosine nucleotides.
In fact, 40 per cent of them should be guanine and 17 per cent cytosine. This is very different from the usual proportions of nucleotides in the same animal; in the DNA of the animal as a whole the proportion of G + C is 39 per cent, and in the ribosomal RNA it is 50 per cent.
After chemical purification of total RNA, a rapidly sedimenting fraction of RNA was isolated by ultracentrifugation which corresponded to expectation, having 40 per cent guanine nucleotides and 19 per cent cytosine nucleotides.
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Furthermore, this particular fraction of RNA is present only in the posterior part of the salivary gland of the silkworm and not in the middle silk gland, which does not produce silk fibroin, nor in the rest of the caterpillar’s body. From this and from the high proportion of G + C, it could be concluded that the isolated RNA was, in fact, messenger RNA for silk fibroin.
Another type of cell which produces only a limited number of proteins at a time is the reticulocyte, which becomes a red blood corpuscle and in the process, synthesizes the two types of polypeptide chains (α and β chains) composing the hemoglobin molecule.
This case differs from the previous one in that the ability of the mRNA to direct protein synthesis was actually tested in living cells. The fraction of RNA presumed to be hemoglobin mRNA was extracted from rabbit reticulocytes and separated from other kinds of RNA by centrifugation on a sucrose gradient.
The preparation, designated as 9s RNA, was injected into frog (Xenopus laevis) oocytes. Oocytes in their growth synthesize large amounts of protein and thus can be expected to have available all the chemical apparatus for protein synthesis, including mRNA of their own.
By supplying the cells with what was expected to be hemoglobin mRNA, it was possible to test whether this foreign mRNA, in combination with other necessary components supplied by the host cells (ribosomes, transfer RNA’s, pool of amino acids, etc.), could cause the synthesis of hemoglobin. Any quantities of hemoglobin discovered in the injected oocytes would be due to the injected mRNA, as oocytes do not normally produce hemoglobin.
In order for the oocytes to synthesize molecules of hemoglobin, and not only blood globin polypeptides, the system had to be supplied with ready-made heme groups, which, not being part of a polypeptide chain, are not directly coded for by mRNA. This requirement was taken care of by injecting some heme molecules in solution simultaneously with the reticulocyte 9s RNA.
The results of the experiment fully justified expectations. A substance could be extracted from the injected oocytes after three or more hours of incubation, which, tested in a variety of ways, short of determining the amino acid sequence, was found to be identical with rabbit hemoglobin.
There appears to be very little doubt that the 9s RNA fraction which was tested was indeed, or contained, the messenger RNA for rabbit hemoglobin. Consequently, it is not possible to interpret the results of the experiment in the sense that the production of hemoglobin was due to some kind of activation of the host cell’s hemoglobin genes.
It is noteworthy that the efficiency of the synthesis in the frog oocytes was several hundred times greater than can be achieved in vitro, in a cell-free system (judged by the amount of protein per amount of RNA), and only several times less than the efficiency of the hemoglobin synthesis in the reticulocytes.
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The frog oocytes were thus shown to be an excellent system for testing mRNA’s, whether fully purified or in a crude form. Following the work of Gurdon’s group, nuclear RNA from frog (Xenopus) neurulae and RNA extracted from ribosomes of young larvae of the same animal were injected into oocytes and caused synthesis of collagen in the oocytes.
This was detected by supplying the oocytes with radioactive hydroxyproline, an amino acid which could only be incorporated into collagen. RNA from gastrulae did not cause collagen to be synthesized. Even though the mRNA for collagen had not been obtained in anything like a pure form, the conclusion may be drawn that mRNA for collagen starts being produced in the neurula stage and not earlier.
In eukaryotes the relation between the gene DNA and the mRNA is far more complicated than in prokaryotes. The “principle of co-linearity” does not hold in eukaryotes. Large quantities of RNA transcribed from the chromosomal DNA accumulate initially within the nucleus.
Some of this RNA has much longer molecules than in the mRNA which eventually is used in protein synthesis, and is in other respects different from the functional mRNA. The intranuclear RNA is generally known as the heterogeneous nuclear RNA—hnRNA.
There is every reason to believe that the hnRNA is a precursor of the functional mRNA which is present in the cytoplasm, but up to 80 per cent of the RNA manufactured in the nucleus never passes into the cytoplasm and is broken down.
At least part of this “rapid turnover nuclear RNA,” however, is directly connected with the production of the RNA’s which later pass into the cytoplasm. Before becoming functional mRNA, elements of the hnRNA have to be processed or transformed in several ways.
One of the causes of differences between hnRNA and the functional mRNA is that in the process of transcription not only the “meaningful” sequences of the genes are transcribed, but also the spacers (introns) and sometimes longer or shorter sequences preceding and following the gene proper are also transcribed.
In some cases more than one gene is transcribed into a single, continuous molecule of RNA, as in the case of the 18S and 28S genes of the rRNA. All this contributes too much greater length of the molecules of the hnRNA than of the mRNA molecules derived from them. The “unnecessary” sequences have to be removed- in the first place, the spacer sequences (introns).
For this purpose the initially produced RNA molecule must be cut at specific points and, then, the “meaningful” parts of the molecule (the exons) have to be rejoined. This is performed presumably by enzyme action within the nucleus after the RNA molecule has been separated from its DNA template.
The mechanism of the cutting up and joining of the RNA molecule is not yet known. Where more than one gene is transcribed into one long molecule of RNA, the sections corresponding to each gene have to be cut out, and any spacer sequences removed. The process of cutting out the unneeded sequences and joining together the meaningful sequences is known as “splicing”.
Even after the “meaningful” part of the RNA molecule has been separated from the “unnecessary” parts, it still has to be further processed. Additional sequences, not transcribed from the gene DNA, are added to the RNA. At the beginning of the sequence (the 5′ end) a modified guanine (7′-methyl guanine) is added.
This is referred to as the “cap,” and the process is known as “capping.” At the end of the RNA molecule there are attached a large number (about 200) of adenosine nucleotides in a process of “adenylation.” The process is presumed to add stability to the mRNA molecule, which, after the process is completed, is designated as poly A+ mRNA.
The mRNA’s for most histones, however, are not adenylated. The adenosine “tail” of the mRNA is not subsequently translated into protein structure. Only after all the above transformations have been performed is the now finally manufactured molecule of RNA ready to leave the nucleus and pass into the cytoplasm via the nuclear pores.