Eukaryotic RNA -- An Introduction to Genetic Analysis -- NCBI Bookshelf

RNA synthesis

Whereas a single RNA polymerase species synthesizes all RNAs in prokaryotes, there are three different RNA polymerases in eukaryotic systems:

1
RNA polymerase I synthesizes rRNA.
2
RNA polymerase II synthesizes mRNA. In eukaryotes, the mRNA molecules always code for one protein, whereas in prokaryotes, many mRNAs code for several proteins.
3
RNA polymerase III synthesizes tRNAs as well as small nuclear and cellular RNA molecules.

The eukaryotic polymerases have a more complex subunit structure than that of prokaryotic polymerases. Some of the subunits are similar to the corresponding E. coli proteins, but others are not.

RNA processing

Figure 10-14
Gene expression in eukaryotes. The mRNA is processed (more...)

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Figure 10-14

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Gene expression in eukaryotes. The mRNA is processed in the nucleus before transport to the cytoplasm. (From J. E. Darnell, Jr., “The Processing of RNA.” Copyright © 1983 by Scientific American, Inc. All rights reserved.)

Figure 10-15
Processing of primary transcript. (a) Transcrip-tion (more...)

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Figure 10-15

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Processing of primary transcript. (a) Transcrip-tion is mediated by RNA polymerase. (b) Early in transcription, an enzyme, guanyltransferase, adds 7-methylguanosine (m⁷Gppp) to the 5′ end of the mRNA. (c) The sequence AAUAAA, near the 3′ end, helps signal a cleavage event (d) by an endonuclease approximately 20 bp farther downstream. (e) An enzyme, poly(A) polymerase, then adds a poly(A) tail, made up of 150 to 200 adenosine residues, to the site of this cleavage at the 3′ end, yielding (f) the complete primary mRNA. (From J. E. Darnell, Jr., “The Processing of RNA.” Copyright © 1983 by Scientific American, Inc. All rights reserved.)

The primary RNA transcript produced in the nucleus is usually processed in several ways before its transport to the cytoplasm, where it is used to program the translation machinery (Figure 10-14

). Figure 10-15

depicts these processing events in detail. First a consisting of a 7-methylguanosine residue linked to the 5′ end of the transcript by a triphosphate bond is added during transcription. Then stretches of adenosine residues are added at the 3′ ends. These poly(A) tails are 150 to 200 residues long. After these modifications, a crucial step removes internal parts of the RNA transcript. The uncovering of this process, and the corresponding realization that genes are “split,” with coding regions interrupted by “intervening sequences,” constitutes one of the most important discoveries in molecular genetics in the past 25 years.

Split genes

Studies of mammalian viral DNA transcripts first suggested a lack of correspondence between the viral DNA and specific mRNA molecules. As recombinant DNA techniques (see Chapter 12) facilitated the physical analysis of eukaryotic genes, it became apparent that primary RNA transcripts were being shortened by the elimination of internal segments before transport into the cytoplasm. In most higher eukaryotes studied, this was found to be true not only for mRNA, but also for rRNA—and even for tRNA in some cases.

Figure 10-16
Split-gene organization of the gene for the protein (more...)

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Figure 10-16

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Split-gene organization of the gene for the protein ovalbumin. (a) The electron micrograph and (b) its map show the result of an experiment in which a single strand of the DNA incorporating the gene for the egg white protein ovalbumin was allowed to hybridize with ovalbumin mRNA, the molecule from which the protein is translated. The looped-out single-stranded segments of DNA represent the introns (c). The schematic representation of the gene shows the seven introns (light green), the eight exons (dark green), and the number of base pairs in each of the exons; the size of the introns ranges from 251 base pairs for intron B to about 1600 (for G). (From P. Chambon, “Split Genes.” Copyright © 1981 by Scientific American, Inc. All rights reserved.)

Figure 10-16

shows the organization of the gene for chicken ovalbumin, a polypeptide consisting of 386 amino acids. The DNA segments that code for the structure of the protein are interrupted by intervening sequences, termed introns. In Figure 10-16

, these segments are designated with the letters A to G. The primary transcript is processed by a series of splicing reactions, much in the same way that a taperecorded message can be cut and pasted back together. Splicing removes the introns and brings together the coding regions, termed exons, to form an mRNA, which now consists of a sequence that is completely colinear with the ovalbumin protein. The exons are indicated by the letter L and numbers 1 to 7 in Figure 10-16

. In different genes, introns have been detected that are as large as 2000 base pairs in length. Some genes have as many as 16 introns.

Figure 10-17
Mature mRNA is produced in a number of steps. (From (more...)

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Figure 10-17

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It is clear that splicing occurs after transcription and in several steps, because RNA transcripts (formerly termed heterogeneous nuclear RNA, or HnRNA) that correspond to the entire genetic region (introns + exons), as well as transcripts intermediate in length, can be isolated. In these intermediate-length RNA molecules, certain introns have already been removed, but others are retained. The entire sequence of events for RNA processing and splicing is summarized in Figure 10-17

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Alternative splicing

Figure 10-18
Complex patterns of eukaryotic mRNA splicing. The (more...)

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Figure 10-18

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Complex patterns of eukaryotic mRNA splicing. The pre-mRNA transcript of the α-tropomyosin gene is alternatively spliced in different cell types. The light green boxes represent introns; the other colors represent exons. Polyadenylation signals are indicated by an A. Dashed lines in the mature mRNAs indicate regions that have been removed by splicing. TM, tropomyosin. (After J. P. Lees et al., Molecular and Cellular Biology 10, 1990, 1729–1742.)

Alternative pathways of splicing can produce different mRNAs and subsequently different proteins from the same primary transcript. The altered forms of the same protein that are generated by alternative splicing are usually used in different cell types or at different stages of development. Figure 10-18

shows the myriad combinations produced by the differential splicing of the primary RNA transcript of the α-tropomyosin gene to ultimately generate a set of related proteins that function optimally in each cell type.

Mechanism of gene splicing

Figure 10-19
Consensus sequences of 5′ and 3′ splice (more...)

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Figure 10-19

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Consensus sequences of 5′ and 3′ splice junctions in eukaryotic mRNAs. Almost all introns begin with GU and end with AG. From the analysis of many exon–intron boundaries, extended consensus sequences of preferred nucleotides at the 5′ and 3′ ends have been established. In addition to AG, other nucleotides just upstream of the 3′ splice junction also are important for precise splicing. (From J. D. Watson, M. Gilman, J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright © 1992 by James D. Watson, Michael Gilman, Jan Witkowski, and Mark Zoller.)

Figure 10-20
Splicing of exons in the primary transcript pre-mRNA (more...)

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Figure 10-20

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Splicing of exons in the primary transcript pre-mRNA takes place in two transesterification reactions. In the first reaction, the ester bond between the 5′ phosphorus of the intron and the 3′ oxygen (red) of exon 1 is exchanged for an ester bond with the 2′ oxygen (dark blue) of the branch-site A residue. In the second reaction, the ester bond between the 5′ phosphorus of exon 2 and the 3′ oxygen (light blue) of the intron is exchanged for an ester bond with the 3′ oxygen of exon 1, releasing the intron as a lariat structure and joining the two exons. Arrows show where the activated hydroxyl oxygens react with phosphorus atoms. (From H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Biology of the Cell, 3d ed. Copyright © 1995 by Scientific American Books, Inc.)

Sequencing of many exon–intron junctions has revealed sequence homologies at these points. As Figure 10-19

shows, a GU is at the 5′ splice site and AG is at the 3′ splice site. It has now been demonstrated that the interaction of small nuclear RNA molecules (snRNAs) interact with the splice site in reactions in which complementary base pairs are coordinated with the splicing enzymes. The splicing reaction itself is diagrammed in Figure 10-20

, which shows an intron being cut out as a branched “lariat” structure, resulting from two successive transesterification reactions. The reactions result in the exchange of one phosphoester bond for another—fusing, or ligating, two exons.

Figure 10-21
Electron micrograph of a spliceosome. (From H. Lodish, (more...)

Figure 10-21

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Electron micrograph of a spliceosome. (From H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology, 3d ed. Copyright © 1995 by Scientific American Books, Inc.)

The sRNAs associate with proteins to form small ribonuclear particles (snRNPs). In higher cells, complexes form between the snRNPs, the primary transcript, and associated factors to form a high-molecular-weight (60S) ribonucleoprotein complex, called a spliceosome (Figure 10-21

), which catalyzes the splicing transesterification reactions.

Self-splicing RNA

Figure 10-22
Splicing mechanisms in group I and group II self-splicing (more...)

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Figure 10-22

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Splicing mechanisms in group I and group II self-splicing introns and spliceosome-catalyzed splicing of pre-mRNA. The intron is shown in blue; the exons to be joined in red. In group I introns (left), a guanosine cofactor (G) associates with the active site. The 3′-hydroxyl group of this guanosine participates in a transesterification reaction with the phosphate at the 5′ end of the intron; this reaction is analogous to that of the 2′-hydroxyl groups of the branch-site A in group II introns and pre-mRNA introns spliced in spliceosomes. The subsequent transesterification that links the 5′ and 3′ exons is similar in all three splicing mechanisms. Note that spliced-out group I introns are linear structures, unlike the branched intron products in the other two cases. (After P. A. Sharp, Science 235, 1987, 769.)

There are now numerous examples of RNA molecules that can catalyze the splicing of their introns without the aid of any proteins. This self-splicing was first shown by Thomas Cech and his co-workers in Tetrahymena and was the first demonstration that an RNA molecule can catalyze a specific biological reaction. These RNAs with enzymatic activity have been termed ribozymes. On the basis of the detailed mechanism of splicing, the introns that are self-spliced are classified as group I or group II introns. The group I introns are found in primary transcripts from some E. coli viruses, Tetrahymena, and certain other single-cell organisms, mitochondria, and chloroplasts, as well as in some tRNAs from bacteria. Group II introns are found in some tRNA primary transcripts and in some chloroplast and mitochondrial primary transcripts. A schematic view of the differences in the splicing mechanism of group I, group II, and spliceosome-dependent introns is shown in Figure 10-22

. The product of the spliced-out group I intron is not a lariat; rather it is a linear molecule.