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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

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RNA Processing and Turnover

Although transcription is the first and most highly regulated step in gene expression, it is usually only the beginning of the series of events required to produce a functional RNA. Most newly synthesized RNAs must be modified in various ways to be converted to their functional forms. Bacterial mRNAs are an exception; as discussed earlier in this chapter, they are used immediately as templates for protein synthesis while still being transcribed. However, the primary transcripts of both rRNAs and tRNAs must undergo a series of processing steps in prokaryotic as well as eukaryotic cells. Primary transcripts of eukaryotic mRNAs similarly undergo extensive modifications, including the removal of introns by splicing, before they are transported from the nucleus to the cytoplasm to serve as templates for protein synthesis. Regulation of these processing steps provides an additional level of control of gene expression, as does regulation of the rates at which different mRNAs are subsequently degraded within the cell.

Processing of Ribosomal and Transfer RNAs

The basic processing of ribosomal and transfer RNAs in prokaryotic and eukaryotic cells is similar, as might be expected given the fundamental roles of these RNAs in protein synthesis. As discussed previously, eukaryotes have four species of ribosomal RNAs (see Table 6.1), three of which (the 28S, 18S, and 5.8S rRNAs) are derived by cleavage of a single long precursor transcript, called a pre-rRNA (Figure 6.37). Prokaryotes have three ribosomal RNAs (23S, 16S, and 5S), which are equivalent to the 28S, 18S, and 5S rRNAs of eukaryotic cells and are also formed by the processing of a single pre-rRNA transcript. The only rRNA that is not processed extensively is the 5S rRNA in eukaryotes, which is transcribed from a separate gene.

Figure 6.37. Processing of ribosomal RNAs.

Figure 6.37

Processing of ribosomal RNAs. Prokaryotic cells contain three rRNAs (16S, 23S, and 5S), which are formed by cleavage of a pre-rRNA transcript. Eukaryotic cells (e.g., human cells) contain four rRNAs. One of these (5S rRNA) is transcribed from a separate (more...)

Prokaryotic and eukaryotic pre-rRNAs are processed in several steps. Initial cleavages of bacterial pre-rRNA yield separate precursors for the three individual rRNAs; these are then further processed by secondary cleavages to the final products. In eukaryotic cells, pre-rRNA is first cleaved at a site adjacent to the 5.8S rRNA on its 5′ side, yielding two separate precursors that contain the 18S and the 28S + 5.8S rRNAs, respectively. Further cleavages then convert these to their final products, with the 5.8S rRNA becoming hydrogen-bonded to the 28S molecule. In addition to these cleavages, rRNA processing involves the addition of methyl groups to the bases and sugar moieties of specific nucleotides, although the function of these modifications is not known.

Like rRNAs, tRNAs in both bacteria and eukaryotes are synthesized as longer precursor molecules (pre-tRNAs), some of which contain several individual tRNA sequences (Figure 6.38). In bacteria, some tRNAs are included in the pre-rRNA transcripts. The processing of the 5′ end of pre-tRNAs involves cleavage by an enzyme called RNase P, which is of special interest because it is a prototypical model of a reaction catalyzed by an RNA enzyme. RNase P consists of RNA and protein molecules, both of which are required for maximal activity. In 1983 Sidney Altman and his colleagues demonstrated that the isolated RNA component of RNase P is itself capable of catalyzing pre-tRNA cleavage. These experiments established that RNase P is a ribozyme—an enzyme in which RNA rather than protein is responsible for catalytic activity.

Figure 6.38. Processing of transfer RNAs.

Figure 6.38

Processing of transfer RNAs. (A) Transfer RNAs are derived from pre-tRNAs, some of which contain several individual tRNA molecules. Cleavage at the 5′ end of the tRNA is catalyzed by the RNase P ribozyme; cleavage at the 3′ end is catalyzed (more...)

The 3′ end of tRNAs is generated by the action of a conventional protein RNase, but the processing of this end of the tRNA molecule also involves an unusual activity: the addition of a CCA terminus. All tRNAs have the sequence CCA at their 3′ ends. This sequence is the site of amino acid attachment, so it is required for tRNA function during protein synthesis. The CCA terminus is encoded in the DNA of some tRNA genes, but in others it is not, instead being added as an RNA processing step by an enzyme that recognizes and adds CCA to the 3′ end of all tRNAs that lack this sequence.

Another unusual aspect of tRNA processing is the extensive modification of bases in tRNA molecules. Approximately 10% of the bases in tRNAs are altered to yield a variety of modified nucleotides at specific positions in tRNA molecules (see Figure 6.38). The functions of most of these modified bases are unknown, but some play important roles in protein synthesis by altering the base-pairing properties of the tRNA molecule (see Chapter 7).

Some pre-tRNAs, as well as pre-rRNAs in a few organisms, contain introns that are removed by splicing. These processing steps are discussed in the next section, together with other splicing reactions.

Processing of mRNA in Eukaryotes

In contrast to the processing of ribosomal and transfer RNAs, the processing of messenger RNAs represents a major difference between prokaryotic and eukaryotic cells. In bacteria, ribosomes have immediate access to mRNA and translation begins on the nascent mRNA chain while transcription is still in progress. In eukaryotes, mRNA synthesized in the nucleus must first be transported to the cytoplasm before it can be used as a template for protein synthesis. Moreover, the initial products of transcription in eukaryotic cells (pre-mRNAs) are extensively modified before export from the nucleus. The processing of pre-mRNA includes modification of both ends of the molecule, as well as the removal of introns from its middle (Figure 6.39).

Figure 6.39. Processing of eukaryotic messenger RNAs.

Figure 6.39

Processing of eukaryotic messenger RNAs. The processing of mRNA involves modification of the 5′ terminus by capping with 7-methylguanosine (m7G), modification of the 3′ terminus by polyadenylation, and removal of introns by splicing. The (more...)

The 5′ end of pre-mRNAs is modified soon after its synthesis by the addition of a structure called a 7-methylguanosine cap. Capping is initiated by the addition of a GTP in reverse orientation to the 5′ terminal nucleotide of the pre-mRNA. Then methyl groups are added to this G residue and to the ribose moieties of one or two 5′ nucleotides of the RNA chain. The 5′ cap aligns eukaryotic mRNAs on the ribosome during translation (see Chapter 7).

The 3′ end of most eukaryotic mRNAs is defined not by termination of transcription, but by cleavage of the primary transcript and addition of a poly-A tail—a processing reaction called polyadenylation (Figure 6.40). Signals for polyadenylation include several sequence elements. The most conserved of these is the hexanucleotide AAUAAA in mammalian cells, which is located 10 to 30 nucleotides upstream of the site of polyadenylation. Less conserved sequences that also contribute to signaling polyadenylation are found both upstream and downstream of the AAUAAA. These sequences are recognized by a complex of proteins, including an endonuclease that cleaves the RNA chain and a separate poly-A polymerase that adds a poly-A tail of about 200 nucleotides to the transcript. The act of polyadenylation signals the termination of transcription, which usually occurs several hundred nucleotides downstream of the site of poly-A addition. The proteins that catalyze polyadenylation are associated with RNA polymerase II, providing a link between transcription and formation of the 3′ end of mRNA.

Figure 6.40. Formation of the 3 ′ ends of eukaryotic mRNAs.

Figure 6.40

Formation of the 3 ′ ends of eukaryotic mRNAs. Polyadenylation signals in mammalian cells consist of the hexanucleotide AAUAAA in addition to upstream and downstream (G-U rich) elements. An endonuclease cleaves the pre-mRNA 10 to 30 nucleotides (more...)

Most mRNAs in eukaryotes are polyadenylated, and poly-A tails have been shown to regulate both translation and mRNA stability. In addition, polyadenylation plays an important regulatory role in early development, where changes in the length of poly-A tails control mRNA translation. For example, many mRNAs are stored in unfertilized eggs in an untranslated form with short poly-A tails (usually 30 to 50 nucleotides long). Fertilization stimulates the lengthening of the poly-A tails of these stored mRNAs, which in turn activates their translation and the synthesis of protiens required for early embryonic development.

The most striking modification of pre-mRNAs is the removal of introns by splicing. As discussed in Chapter 4, the coding sequences of most eukaryotic genes are interrupted by noncoding sequences (introns) that are precisely excised from the mature mRNA. Most genes contain multiple introns, which typically account for about ten times more pre-mRNA sequences than the exons do. The unexpected discovery of introns in 1977 generated an active research effort directed toward understanding the mechanism of splicing, which had to be highly specific to yield functional mRNAs. Further studies of splicing have not only illuminated new mechanisms of gene regulation; they have also revealed novel catalytic activities of RNA molecules.

Splicing Mechanisms

The key to understanding pre-mRNA splicing was the development of in vitro systems that efficiently carried out the splicing reaction (Figure 6.41). Pre-mRNAs were synthesized in vitro by the cloning of structural genes (with their introns) adjacent to promoters for bacteriophage RNA polymerases, which could readily be isolated in large quantities. Transcription of these plasmids could then be used to prepare large amounts of pre-mRNAs that, when added to nuclear extracts of mammalian cells, were found to be correctly spliced. As with transcription, the use of such in vitro systems has allowed splicing to be analyzed in much greater detail than would have been possible in intact cells.

Figure 6.41. In vitro splicing.

Figure 6.41

In vitro splicing. A gene containing an intron is cloned downstream of a promoter (P) recognized by a bacteriophage RNA polymerase. The plasmid is digested with a restriction enzyme that cleaves at the 3′ end of the inserted gene to yield a linear (more...)

Analysis of the reaction products and intermediates formed in vitro revealed that pre-mRNA splicing proceeds in two steps (Figure 6.42). First, the pre-mRNA is cleaved at the 5′ splice site, and the 5′ end of the intron is joined to an adenine nucleotide within the intron (near its 3′ end). In this step an unusual bond forms between the 5′ end of the intron and the 2′ hydroxyl group of the adenine nucleotide. The resulting intermediate is a lariat-like structure, in which the intron forms a loop. The second step in splicing then proceeds with simultaneous cleavage at the 3′ splice site and ligation of the two exons. The intron is thus excised as a lariat-like structure, which is then linearized and degraded within the nucleus of intact cells.

Figure 6.42. Splicing of pre-mRNA.

Figure 6.42

Splicing of pre-mRNA. The splicing reaction proceeds in two steps. The first step involves cleavage at the 5′ splice site (SS) and joining of the 5′ end of the intron to an A within the intron (the branch point). This reaction yields a (more...)

These reactions define three critical sequence elements of pre-mRNAs: sequences at the 5′ splice site, sequences at the 3′ splice site, and sequences within the intron at the branch point (the point at which the 5′ end of the intron becomes ligated to form the lariat-like structure) (see Figure 6.42). Pre-mRNAs contain similar consensus sequences at each of these positions, allowing the splicing apparatus to recognize pre-mRNAs and carry out the cleavage and ligation reactions involved in the splicing process.

Biochemical analysis of nuclear extracts has revealed that splicing takes place in large complexes, called spliceosomes, composed of proteins and RNAs. The RNA components of the spliceosome are five types of small nuclear RNAs (snRNAs) called U1, U2, U4, U5, and U6. These snRNAs, which range in size from approximately 50 to nearly 200 nucleotides, are complexed with six to ten protein molecules to form small nuclear ribonucleoprotein particles (snRNPs), which play central roles in the splicing process. The U1, U2, and U5 snRNPs each contain a single snRNA molecule, whereas U4 and U6 snRNAs are complexed to each other in a single snRNP.

The first step in spliceosome assembly is the binding of U1 snRNP to the 5′ splice site of pre-mRNA (Figure 6.43). This recognition of 5′ splice sites involves base pairing between the 5′ splice site consensus sequence and a complementary sequence at the 5′ end of U1 snRNA (Figure 6.44). U2 snRNP then binds to the branch point, by similar complementary base pairing between U2 snRNA and branch point sequences. A preformed complex consisting of U4/U6 and U5 snRNPs is then incorporated into the spliceosome, with U5 binding to sequences upstream of the 5′ splice site. The splicing reaction is then accompanied by rearrangements of the snRNAs. Prior to the first reaction step (formation of the lariat-like intermediate, see Figure 6.42), U6 dissociates from U4 and displaces U1 at the 5′ splice site. U5 then binds to sequences at the 3′ splice site, followed by excision of the intron and ligation of the exons.

Figure 6.43. Assembly of the spliceosome.

Figure 6.43

Assembly of the spliceosome. The first step in spliceosome assembly is the binding of U1 snRNP to the 5′ splice site (SS), followed by the binding of U2 snRNP to the branch point. A preformed complex consisting of U4/U6 and U5 snRNPs then enters (more...)

Figure 6.44. Binding of U1 snRNA to the 5′ splice site.

Figure 6.44

Binding of U1 snRNA to the 5′ splice site. The 5′ terminus of U1 snRNA binds to consensus sequences at 5′ splice sites by complementary base pairing.

Not only do the snRNAs recognize consensus sequences at the branch points and splice sites of pre-mRNAs; they also catalyze the splicing reaction directly. The catalytic role of RNAs in splicing was demonstrated by the discovery that some RNAs are capable of self-splicing; that is, they can catalyze the removal of their own introns in the absence of other protein or RNA factors. Self-splicing was first described by Tom Cech and his colleagues during studies of the 28S rRNA of the protozoan Tetrahymena. This RNA contains an intron of approximately 400 bases that is precisely removed following incubation of the pre-rRNA in the absence of added proteins. Further studies have revealed that splicing is catalyzed by the intron, which acts as a ribozyme to direct its own excision from the pre-rRNA molecule. The discovery of self-splicing of Tetrahymena rRNA, together with the studies of RNase P already discussed, provided the first demonstrations of the catalytic activity of RNA.

Additional studies have revealed self-splicing RNAs in mitochondria, chloroplasts, and bacteria. These self-splicing RNAs are divided into two classes on the basis of their reaction mechanisms (Figure 6.45). The first step in splicing for group I introns (e.g., Tetrahymena pre-rRNA) is cleavage at the 5′ splice site mediated by a guanosine cofactor. The 3′ end of the free exon then reacts with the 3′ splice site to excise the intron as a linear RNA. In contrast, the self-splicing reactions of group II introns (e.g., some mitochondrial pre-mRNAs) closely resemble those characteristic of nuclear pre-mRNA splicing, in which cleavage of the 5′ splice site results from attack by an adenosine nucleotide in the intron. As with pre-mRNA splicing, the result is a lariat-like intermediate, which is then excised.

Figure 6.45. Self-splicing introns.

Figure 6.45

Self-splicing introns. Group I and group II self-splicing introns are distinguished by their reaction mechanisms. In group I introns, the first step in splicing is cleavage of the 5′ splice site by reaction with a guanosine cofactor. The result (more...)

The similarity between spliceosome-mediated pre-mRNA splicing and self-splicing of group II introns strongly suggested that the active catalytic components of the spliceosome were RNAs rather than proteins. In particular, these similarities suggested that pre-mRNA splicing was catalyzed by the snRNAs of the spliceosome. Continuing studies of pre-mRNA splicing have provided clear support for this view; U2, U5, and U6 snRNAs have been identified as catalytic components of the spliceosome. Pre-mRNA splicing is thus considered to be an RNA-based reaction, catalyzed by spliceosome snRNAs acting analogously to group II self-splicing introns. Protein components of the snRNPs are also required, however, and participate in both assembly of the spliceosome and the splicing reaction. In addition, a number of proteins that are not snRNP components play auxiliary roles in splicing and spliceosome assembly. One important role of both snRNP and non-snRNP proteins is to identify and select the splice sites that are recognized by the snRNAs. For example, protein splicing factors are required for the binding of both U1 and U2 snRNPs to the appropriate sites on pre-mRNA. Because many pre-mRNAs contain multiple introns, the splicing machinery must be able to identify and join the appropriate 5′ and 3′ splice sites to produce a functional mRNA. The mechanism of such splice site selection is not understood, but protein splicing factors clearly play an important role.

Finally, it should be noted that the mechanism of pre-tRNA splicing differs from both types of self-splicing reactions and from the splicing of nuclear pre-mRNA. Pre-tRNA splicing involves conventional protein enzymes with no known role for RNA catalysis. Instead, a protein endonuclease cleaves at the splice sites, excising the intron as a linear RNA. This step is followed by ligation of the exons, yielding the mature tRNA molecule.

Alternative Splicing

The central role of splicing in the processing of pre-mRNA opens the possibility of regulation of gene expression by control of the activity of the splicing machinery. Moreover, since most pre-mRNAs contain multiple introns, different mRNAs can be produced from the same gene by different combinations of 5′ and 3′ splice sites. The possibility of joining exons in varied combinations provides a novel means of controlling gene expression by generating multiple mRNAs (and therefore multiple proteins) from the same pre-mRNA. This process, called alternative splicing, occurs frequently in genes of complex eukaryotes and provides an important mechanism for tissue-specific and developmental regulation of gene expression.

One interesting example of alternative splicing is provided by some genes that encode transcriptional regulatory proteins. In several cases, alternative splicing of these pre-mRNAs yields products with dramatically different functions—namely, the ability to act as either activators or repressors of transcription (Figure 6.46). As discussed earlier, transcriptional activators consist of two distinct domains: a DNA-binding domain and an activation domain. These domains are generally encoded in separate exons, so alternative splicing allows them to be reassorted into different combinations, thereby enabling the production of activators and repressors from the same gene. Splicing that yields an mRNA that contains exons encoding both DNA-binding and activation domains will result in synthesis of an activator protein. Alternative splicing, however, may result in the formation of an mRNA encoding a DNA-binding domain but lacking an activation domain. Translation of such an alternatively spliced mRNA will result in synthesis of a repressor, which will suppress gene expression by competing with the activator for binding to target DNA sequences (see Figure 6.30).

Figure 6.46. Alternative splicing of pre-mRNA that encodes a transcription factor.

Figure 6.46

Alternative splicing of pre-mRNA that encodes a transcription factor. In this example, a transcriptional regulatory protein is encoded by four exons: the first encodes the DNA-binding domain; the third encodes the activation domain. The pre-mRNA is subject (more...)

Because patterns of alternative splicing can vary in different tissues, regulation of splicing provides an important means of regulating tissue-specific gene expression. Although the mechanism by which the correct splice sites are selected in a pre-mRNA is not known, several protein factors have been identified that contribute to splice site selection and can affect the use of alternative splice sites in a pre-mRNA molecule. Variations in the expression of such splicing factors in different cell types may result in tissue-specific patterns of alternative splicing, thereby contributing to the regulation of gene expression during development and differentiation. A particularly notable example is provided by sex determination in Drosophila, where alternative splicing of the same pre-mRNA determines whether a fly is male or female.

RNA Editing

RNA editing refers to RNA processing events (other than splicing) that alter the protein-coding sequences of some mRNAs. This unexpected form of RNA processing was first discovered in mitochondrial mRNAs of trypanosomes, in which U residues are added and deleted at multiple sites along the molecule. More recently, editing has also been described in mitochondrial mRNAs of other organisms, chloroplast mRNAs of higher plants, and nuclear mRNAs of some mammalian genes.

Editing in mammalian nuclear mRNAs, as well as in mitochondrial and chloroplast RNAs of higher plants, involves single base changes as a result of base modification reactions, similar to those involved in tRNA processing. In mammalian cells, RNA editing reactions include the deamination of cytosine to uridine and of adenosine to inosine. One of the best-studied examples is editing of the mRNA for apolipoprotein B, which transports lipids in the blood. In this case, tissue-specific RNA editing results in two different forms of apolipoprotein B (Figure 6.47). In humans, Apo-B100 (4536 amino acids) is synthesized in the liver by translation of the unedited mRNA. However, a shorter protein (Apo-B48, 2152 amino acids) is synthesized in the intestine as a result of translation of an edited mRNA in which a C has been changed to a U by deamination. This alteration changes the codon for glutamine (CAA) in the unedited mRNA to a translation termination codon (UAA) in the edited mRNA, resulting in synthesis of the shorter Apo-B protein. Tissue-specific editing of Apo-B mRNA thus results in the expression of structurally and functionally different proteins in liver and intestine. The full-length Apo-B100 produced by the liver transports lipids in the circulation; Apo-B48 functions in the absorption of dietary lipids by the intestine.

Figure 6.47. Editing of apolipoprotein B mRNA.

Figure 6.47

Editing of apolipoprotein B mRNA. In human liver, unedited mRNA is translated to yield a 4536-amino-acid protein called Apo-B100. In human intestine, however, the mRNA is edited by a base modification that changes a specific C to a U. This modification (more...)

RNA Degradation

The processing steps discussed in the previous section result in the formation of mature mRNAs, which then direct protein synthesis. However, what may be considered the final aspect of the processing of an RNA molecule is its eventual degradation within the cell. Since the intracellular level of any RNA is determined by a balance between synthesis and degradation, the rate at which individual RNAs are degraded is another level at which gene expression can be controlled. Both ribosomal and transfer RNAs are very stable, and this stability largely accounts for the high levels of these RNAs (greater than 90% of all RNA) in both prokaryotic and eukaryotic cells. In contrast, bacterial mRNAs are rapidly degraded, usually having half-lives of only 2 to 3 minutes. This rapid turnover of bacterial mRNAs allows the cell to respond quickly to alterations in its environment, such as changes in the availability of nutrients required for growth. In eukaryotic cells, however, different mRNAs are degraded at different rates, providing an additional parameter to the regulation of eukaryotic gene expression.

The degradation of most eukaryotic mRNAs is initiated by shortening of their poly-A tails. Then follows removal of the 5′ cap and degradation of the RNA by nucleases acting from both ends. The half-lives of mRNAs in mammalian cells vary from less than 30 minutes to approximately 20 hours. The unstable mRNAs frequently code for regulatory proteins, including certain transcription factors, whose levels within the cell vary rapidly in response to environmental stimuli. These mRNAs often contain specific AU-rich sequences near their 3′ ends that appear to signal rapid degradation by promoting deadenylation.

The stability of some mRNAs can also be regulated in response to extracellular signals. A good example is provided by the mRNA that encodes transferrin receptor—a cell surface protein involved in the uptake of iron by mammalian cells. The amount of transferrin receptor within cells is regulated by the availability of iron, largely as a result of modulation of the stability of its mRNA (Figure 6.48). In the presence of adequate amounts of iron, transferrin receptor mRNA is rapidly degraded as a result of specific nuclease cleavage at a sequence near its 3′ end. If an adequate supply of iron is not available, however, the mRNA is stabilized, resulting in increased synthesis of transferrin receptor and more iron uptake by the cell. This regulation is mediated by a protein that binds to specific sequences (called the iron response element, or IRE) near the 3′ end of transferrin receptor mRNA and protects the mRNA from cleavage. The binding of this regulatory protein to the IRE is in turn controlled by the levels of iron within the cell: If iron is scarce, the protein binds to the IRE and protects transferrin receptor mRNA from degradation. Similar changes in the stability of other mRNAs are involved in the regulation of gene expression by certain hormones. Thus, although transcription remains the primary level at which gene expression is regulated, variations in the rate of mRNA degradation also play an important role in controlling steady-state levels of mRNAs within the cell.

Figure 6.48. Regulation of transferrin receptor mRNA stability.

Figure 6.48

Regulation of transferrin receptor mRNA stability. The levels of transferrin receptor mRNA are regulated by the availability of iron. If the supply of iron is adequate, the mRNA is rapidly degraded as a result of nuclease cleavage near the 3′ (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, Geoffrey M Cooper.
Bookshelf ID: NBK9864

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