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HOME :: CHAPTER 5 :: DIFFERENTIAL NRNA CENSORING :: COMPLEXITY AND HETEROGENEOUS NUCLEAR RNA |
Complexity and Heterogeneous Nuclear RNA
During the 1960s, developmental biologists had a strong bias toward explaining differentiation in terms of transcriptional regulation of gene expression. First, research on polytene chromosomes and their products indicated that transcriptional regulation occurs during insect development. Second, DNA-RNA hybridization studies showed that whereas the DNA of different cell types is identical, their mRNAs are different (McCarthy and Hoyer, 1964). Third, gene expression could be studied more readily in bacteria than in eukaryotic cells, and this research demonstrated the importance of transcriptional regulation in producing different physiological states in Escherichia coli. The lac operon of E. coli, for instance, transcribes mRNA only in the presence of a specific inducer molecule (in this case, lactose). Transcriptional gene expression in unicellular prokaryotes was a powerful paradigm for differential gene expression in developing eukaryotic cells.
Discovery of a primary nuclear transcript, heterogeneous nuclear RNA
It soon became apparent, however, that messenger RNA is not the primary transcription product of eukaryotic cells. Rather, there is nuclear RNA that was much larger than mRNA and has a much shorter half-life than the cytoplasmic messages. Because they vary widely in size, these nuclear RNA molecules are called heterogeneous nuclear RNA (hnRNA). The hnRNA molecules are approximately 5 to 20 X 103 nucleotides long, compared with the 2 X 103 bases usually found in mRNA, and they decay with a half-life of minutes instead of hours (see Lewin, 1980). In sea urchin embryos, the half-life of mRNA is about 5 hours whereas the half-life of nuclear RNA is about 20 minutes. The relationship between hnRNA and cytoplasmic mRNA was disputed until 1977, at which time several laboratories found mRNA sequences within the large nuclear RNA molecules. Bastos and Aviv (1977) isolated mouse globin message and made complementary DNA from it by using reverse transcriptase (Figure 1). They then conjugated this cDNA to cellulose beads. After nucleated hemoglobin-synthesizing cells were grown with radioactive uridine, the radiolabeled RNA was extracted and passed over this column. Any RNA sequence coding for globin should bind to the complementary DNA on the column. No other RNA sequence should be "fished out" in such a manner. The RNA bound to the column was eluted from the column by washing it with low-ionic-strength solutions, which destabilized the hydrogen bonds holding the complementary molecules together. When cells were labeled for 5 minutes, the radioactive RNA binding to the column had an average size of 5000 nucleotides, nearly eight times that of the cytoplasmic globin message.
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| Figure 1 Protocol for isolating heterogeneous nuclear RNA containing globin-coding sequences. |
However, when the labeling was stopped and the RNA extracted 5 minutes later, the globin sequences were found in a smaller RNA. Finally, the globin-coding RNA was found in a 600-nucleotide molecule characteristic of the cytoplasmic message. Thus, hnRNA appears to contain the precursors of cytoplasmic globin mRNA. Furthermore, studies of Beta+-thalassemia, a disease characterized by low levels of Beta-globin, showed that the basis of this disease is the faulty processing and transport of the globin message precursor inside the nucleus (Kantor et al., 1980; Marquat et al., 1980). Thus, at least some of the hnRNA contains precursor mRNA.
The next question involved the number of different types of RNA sequences found in the nucleus and cytoplasm. Does the nucleus contain a greater diversity of sequences than the cytoplasm, or are they about the same? The answer to this question will tell us whether much more of the genome is transcribed into hnRNA than the amount we measure in mRNA. The diversity of nucleotide sequences is called complexity. If one were to take one copy of each different mRNA and lay them end to end, and express the total length in nucleotides, that is the complexity of the mRNA.
The analysis of complexity is often graphed on C0t curves. C0 refers to the original concentration of labeled nucleic acid (in moles of nucleotide per liter) and t refers to the time (in seconds). The extent of reassociation is a function of both concentration and time. Thus, the percentage of renatured DNA is plotted against the C0t value (Figure 2). This type of analysis has revealed that the genome contains some DNA sequences represented millions of times (the highly repetitive fraction), some sequences represented thousands of times (the moderately repetitive fraction), and some DNA (such as those genes coding for enzymes) that are essentially represented once per haploid genome (Figure 2B). This last fraction is referred to as single-copy DNA (Britten and Kohne, 1968).
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| Figure 2 Reassociation of nucleic acids from various sources. The curve represents the normal kinetics expected when each gene is present in the same frequency. Because the hybridization reaction demands that two complementary sequences come together, the rate of hybridization can be described as dC/dt = -kC2, where C is the concentration of single-stranded sequences present at time t, and k is the association constant. Note that the ordinate is plotted with 0 percent at the top and that the C0t axis is represented logarithmically. (A) Reassociation of DNAs or artificial RNA with little or no repetitive sequences. According to these data, the DNA of E. coli has about 100 times as many sequences as the DNA of the T4 bacteriophage has. (B) Reassociation of DNA from a typical eukaryotic organism. DNA is sheared to about 500 nucleotide pairs and denatured in alkali. When the DNA is allowed to renature, a fraction of highly repetitive DNA reanneals almost immediately. Then a fraction of moderately repetitive DNA renatures. Eventually the "single-copy" region of the genomethe portion that encodes the proteinsrenatures. According to these data, some sequences are present a million times per haploid genome, whereas most sequences are present only once. (A after Britten and Kohne, 1968; B after Hood et al., 1975.) |
To measure complexity of RNA, RNA is isolated from the tissue of interest and a small amount of radioactive single-copy DNA is added to a great abundance of the RNA. The fraction of DNA that finds its "partner" is the fraction of the genome that is transcribed into RNA (after being multiplied by 2 to account for the presence of both strands in the single-copy DNA). By this method the hnRNA complexity was determined to be more than tenfold greater than that of mRNA from the same cells. About 90 percent of the hnRNA contains sequences that do not enter the cytoplasm.
Literature Cited
Bastos, R. N. and Aviv, H. 1977. Globin RNA precursor molecules: Biosynthesis and processing erythroid cells. Cell 11: 641-650.
Britten, R. J. and Kohne, D. E. 1968. Repeated sequences in DNA. Science 161: 529-540.
Kantor, J. A., Turner, P. H. and Nienhuis, A. W. 1980. β Thalassemia: Mutations which affect processing of the β-globin mRNA precursor. Cell 21: 149-157.
Lewin, B. 1980. Gene Expression 2, 2nd Ed. Wiley-Interscience, New York, pp. 694-760.
Maquat, L. E., Kinniburgh, A. J., Beach, L. R., Honig, G. R., Lazerson, J., Ershler, W. B. and Ross, J. 1980. Processing of human β-globin mRNA precursor to mRNA is defective in three patients with β+ thalassemias. Proc. Natl. Acad. Sci. USA 77: 4287-4291.
McCarthy, B. J. and Hoyer, B. H. 1964. Identity of DNA and diversity of RNA in normal mouse tissues. Proc. Natl. Acad. Sci. USA 52: 915-922.
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HOME :: CHAPTER 5 :: DIFFERENTIAL NRNA CENSORING :: COMPLEXITY AND HETEROGENEOUS NUCLEAR RNA |