Now that we’ve seen how pre-mRNAs are processed into mature, functional mRNAs, let’s consider how regulation of this process can contribute to gene control. Recall from Chapter 8 that higher eukaryotes have both simple and com plex transcription units encoded in their DNA. The primary transcripts produced from the former contain one poly(A) site and exhibit only one pattern of RNA splicing, even if multiple introns are present; thus simple transcription units encode a single mRNA. In contrast, the primary transcripts produced from complex transcription units (which constitute about 95 percent of all human transcription units) can be processed in alternative ways to yield different mRNAs that encode distinct proteins.

Alternative Splicing Generates Transcripts with Different Combinations of Exons

The discovery that a large fraction of transcription units in higher organisms encode alternatively spliced mRNAs and that differently spliced mRNAs are expressed in different cell types revealed that regulation of RNA splicing is an important gene-control mechanism in higher eukaryotes. Although many examples of cleavage at alternative poly(A) sites in pre-mRNAs are known, alternative splicing of different exons is the more common mechanism for expressing different proteins from one complex transcription unit. In Chapter 5, for example, we mentioned that fibroblasts produce one type of the extracellular protein fibronectin, whereas hepatocytes produce another type. Both fibronectin isoforms are encoded by the same transcription unit, but the transcript is spliced differently in the two cell types to yield two different mRNAs. In other cases, alternative processing of the same transcript may occur simultaneously in the same cell type in response to different developmental or environmental signals. We first discuss one of the best-understood examples of regulated RNA processing, then briefly consider the consequences of RNA splicing in the development of the nervous system.

 A Cascade of Regulated RNA Splicing Controls Drosophila Sexual Differentiation

One of the earliest examples of regulated alternative splicing of pre-mRNA came from studies of sexual differentiation in Drosophila. The genes required for normal Drosophila sexual differentiation were first characterized by isolating Drosophila mutants defective in the process. When the proteins encoded by the wild-type genes were characterized biochemically, two of them were found to regulate a cascade of alternative RNA splicing in Drosophila embryos. More recent research has provided insight into how these proteins regulate RNA processing and ultimately lead to the creation of two different sex-specific transcriptional repressors that suppress the development of characteristics of the opposite sex.

The Sex-lethal (Sxl) protein, encoded by the sex-lethal gene, is the first protein to act in the cascade (Figure 1). The Sxl protein is present only in female embryos. Early in development, the Sxl gene is transcribed from a promoter that functions only in female embryos. Later in development, this female-specific promoter is shut off, and another promoter for sex-lethal becomes active in both male and female embryos. In male embryos, however, in the absence of early Sxl protein, exon 2 of the sex-lethal pre-mRNA is spliced to exon 3 to produce an mRNA that contains a stop codon early in the sequence. The net result is that male embryos produce no functional Sxl protein either early or later in development.

Fig 1. A cascade of regulated splicing controls sex determination in Drosophila embryos. For clarity, only the exons (boxes) and introns (black lines) where regulated splicing occurs are shown. Splicing is indicated by red dashed lines above (female) and blue dashed lines below (male) the pre-mRNAs. Vertical red lines in exons indicate in-frame stop codons, which prevent synthesis of functional protein. Only female embryos produce functional Sxl protein, which represses splicing between exons 2 and 3 in sxl pre-mRNA (a) and between exons 1 and 2 in tra pre-mRNA (b). (c) In contrast, the cooperative binding of Tra protein and two SR proteins, Rbp1 and Tra2, activates splicing between exons 3 and 4 and cleavage/polyadenylation(An) at the 3′ end of exon 4 in dsx pre-mRNA in female embryos. In male embryos, which lack functional Tra, the SR proteins do not bind to exon 4, and consequently exon 3 is spliced to exon 5. The distinct Dsx proteins produced in female and male embryos as the result of this cascade of regulated splicing repress transcription of genes required for sexual differentiation of the opposite sex. See M. J. Moore et al., 1993, in R. Gesteland and J. Atkins, eds., The RNA World, Cold Spring Harbor Press, pp. 303–357.

In contrast, the Sxl protein expressed in early female embryos regulates splicing of the sex-lethal pre-mRNA so that a functional sex-lethal mRNA is produced (Figure 1a). Sxl accomplishes this by binding to a sequence in the pre-mRNA near the 3′ end of the intron between exon 2 and exon 3, thereby blocking the proper association of U2AF and U2 snRNP with the adjacent 3′ splice site used in males. As a consequence, the U1 snRNP bound to the 5′ end of the intron between exons 2 and 3 assembles into a spliceosome with U2 snRNP bound to the branch point at the 3′ end of the intron between exons 3 and 4, leading to the splicing of exon 2 to exon 4 and the skipping of exon 3. The binding site for Sxl in the sex-lethal pre-mRNA is called an intronic splicing silencer because of its location in an intron and its function in blocking, or “silencing,” the use of a splice site. The resulting female-specific sex-lethal mRNA is translated into functional Sxl protein, which rein forces its own expression in female embryos by continuing to cause skipping of exon 3. The absence of Sxl protein in male embryos allows the inclusion of exon 3 and, consequently, of the stop codon near the 5′ end of exon 3 that prevents translation of functional Sxl protein (see Figure 1a).

Sxl protein also regulates alternative splicing of the pre-mRNA transcribed from the transformer gene (Figure 1b). In male embryos, in which no Sxl is ex pressed, exon 1 is spliced to exon 2, which contains a stop codon that prevents synthesis of a functional Transformer (Tra) protein. In female embryos, however, binding of Sxl protein to an intronic splicing silencer at the 3′ end of the intron between exons 1 and 2 blocks binding of U2AF at this site. The interaction of Sxl with transformer pre-mRNA is mediated by two adjacent RRM domains in the protein. When Sxl is bound, U2AF binds to a lower-affinity site farther 3′ in the pre-mRNA; as a result, exon 1 is spliced to this alternative 3′ splice site, causing skipping of exon 2 with its stop codon. The resulting female-specific transformer mRNA, which contains additional constitutively spliced exons, is translated into functional Tra protein.

Finally, Tra protein regulates the alternative processing of pre-mRNA transcribed from the doublesex (dsx) gene (Figure 1c). In female embryos, a complex of Tra and two constitutively expressed SR proteins, Rbp1 and Tra2, directs the splicing of exon 3 to exon 4 and also promotes cleavage/polyadenylation at the alternative poly(A) site at the 3′ end of exon 4, leading to a short, female-specific version of the Dsx protein. In male embryos, which produce no Tra protein, exon 4 is skipped, so that exon 3 is spliced to exon 5. Exon 5 is constitutively spliced to exon 6, which is polyadenylated at its 3′ end—leading to a longer, male-specific version of the Dsx protein. The RNA sequence to which Tra binds in exon 4 is called an exonic splicing enhancer because it enhances splicing at a nearby splice site.

As a result of the cascade of regulated RNA processing depicted in Figure 10-18, different Dsx proteins are ex pressed in male and female embryos. The two proteins are transcription factors that share the N-terminal sequence encoded in exons 1–3, including a common DNA-binding domain, but have different C-terminal sequences, encoded by exon 4 in females and exon 5 plus additional downstream exons in males. The unique C-terminal end of the female protein functions as a strong activation domain, while the C-terminal end of the male protein is a strong repression domain. Consequently, the female Dsx protein activates genes with binding sites for the transcription factor, including genes that induce development of female characteristics, while the male Dsx protein represses the same target genes.

Figure 2 illustrates how the Tra/Tra2/Rbp1 complex is thought to interact with doublesex pre-mRNA. Rbp1 and Tra2 are SR proteins, but they do not interact with exon 4 in the absence of the Tra protein. The interaction of the Tra protein with Rbp1 and Tra2 results in the cooperative binding of all three proteins to six exonic splicing enhancers in exon 4. The bound Tra2 and Rbp1 proteins then promote the binding of U2AF and the U2 snRNP to the 3′ end of the intron between exons 3 and 4, just as other SR proteins do for constitutively spliced exons. The Tra/ Tra2/Rbp1 complexes also enhance binding of the cleavage/ polyadenylation complex to the 3′ end of exon 4 because the U2 snRNP plus associated proteins bound to a 3′ splice site enhance binding of cleavage/polyadenylation factors to an appropriately spaced polyadenylation signal through cooperative binding interactions.

Fig2. Model of splicing activation by Tra protein and the SR proteins Rbp1 and Tra2. In female Drosophila embryos, splicing of exons 3 and 4 in dsx pre-mRNA is activated by the binding of Tra/Tra2/Rbp1 complexes to six exonic splicing enhancers in exon 4. Because Rbp1 and Tra2 cannot bind to the pre-mRNA in the absence of Tra, exon 4 is skipped in male embryos. See the text for discussion. An = polyadenylation. See T. Maniatis and B. Tasic, 2002, Nature 418:236.

Splicing Repressors and Activators Control Splicing at Alternative Sites

As is evident from Figure 1, the Drosophila Sxl protein and Tra protein have opposite effects: Sxl prevents splicing, causing exons to be skipped, whereas Tra promotes splicing. The action of similar proteins may explain the cell-type-specific expression of fibronectin isoforms in humans. For instance, an Sxl-like splicing repressor expressed in hepatocytes might bind to splice sites for the EIIIA and EIIIB exons in the fibronectin pre-mRNA, causing them to be skipped during RNA splicing. Alternatively, a Tra-like splicing activator expressed in fibroblasts might activate the splice sites associated with those exons, leading to their inclusion in the mature mRNA. Experimental examination of some systems has revealed that the inclusion of an exon in some cell types and the skipping of the same exon in other cell types results from the combined influence of several splicing repressors (usually hnRNP proteins) and enhancers (usually SR proteins). RNA binding sites for repressors can also occur in exons, where they are called exonic splicing silencers. And binding sites for splicing activators can also occur in introns, where they are called intronic splicing enhancers.

Alternative splicing of exons is especially common in the nervous system, where it generates multiple isoforms of many proteins required for neuronal development and function in both vertebrates and invertebrates. The primary transcripts of the genes encoding these proteins often show complex splicing patterns that can generate several different mRNAs, which are expressed in different anatomic locations within the central nervous system. Here we consider two remarkable examples that illustrate the critical role of this process in neural function.

Expression of K+-Channel Proteins in Vertebrate Hair Cells In the inner ear of vertebrates, individual hair cells, which are ciliated neurons, respond most strongly to a specific frequency of sound. Cells tuned to low frequencies (~50 Hz) are found at one end of the tubular cochlea that makes up the inner ear; cells responding to high frequencies (~5000 Hz) are found at the other end (Figure 3a). Cells in between the two ends respond to a gradient of frequencies between these extremes. One component in the tuning of hair cells in reptiles and birds is the opening of K+ ion channels in response to increased intracellular Ca2+ concentrations. The Ca2+ concentration at which the channel opens determines the frequency with which the membrane potential oscillates and hence the frequency to which the cell is tuned.

Fig3. Role of alternative splicing in the perception of sounds of different frequencies. (a) The chicken cochlea, a 5-mm long tube, contains an epithelium of auditory hair cells that are tuned to a gradient of vibrational frequencies from 50 Hz at the apical end (left) to 5000 Hz at the basal end (right). (b) The Ca2+-activated K+ channel contains seven transmembrane α helices (S0–S6), which associate to form the channel. The cytosolic domain, which includes four hydrophobic regions (S7–S10), regulates opening of the channel in response to Ca2+. Isoforms of the channel, encoded by alternatively spliced mRNAs produced from the same primary transcript, open at different Ca2+ concentrations and thus respond to different frequencies. Red numbers refer to regions where alternative splicing produces different amino acid sequences in the various isoforms. See K. P. Rosenblatt et al., 1997, Neuron 19:1061.

The gene encoding this Ca2+-activated K+ channel is ex pressed as multiple, alternatively spliced mRNAs, which encode proteins that open at different Ca2+ concentrations. Hair cells with different response frequencies express different iso forms of the channel protein depending on their position along the length of the cochlea. The sequence variation in the protein is very complex: there are at least eight regions in the mRNA where one of several alternative exons is utilized, permitting the expression of 576 possible isoforms (Figure 3b).

PCR analysis of mRNAs from individual hair cells has shown that each hair cell expresses a mixture of different K+-channel mRNAs, with different isoforms predominating in different cells according to their position along the cochlea. This remarkable arrangement suggests that splicing of the K+-channel pre-mRNA is regulated in response to extracellular signals that inform the cell of its position along the cochlea.

Other studies have demonstrated that splicing at one of the alternative splice sites in the Ca2+-activated K+-channel pre-mRNA in the rat is suppressed when a specific protein kinase is activated by neuron depolarization in response to synaptic activity from interacting neurons. This observation raises the possibility that a splicing repressor specific for this splice site may be activated when it is phosphorylated by this protein kinase, whose activity in turn is regulated by synaptic activity. Since hnRNP and SR proteins are extensively modified by phosphorylation and other post-translational modifications, it seems likely that complex regulation of alternative RNA splicing through post-translational modifications of splicing factors plays a significant role in modulating neuron function.

Many examples of genes similar to those that encode the cochlear K+ channel have been observed in vertebrate neurons; in these cases, alternatively spliced mRNAs co-expressed from a specific gene in one type of neuron are expressed at different relative concentrations in different regions of the central nervous system. Expansions in the number of microsatellite repeats within the transcribed regions of genes expressed in neurons can alter the relative concentrations of alternatively spliced mRNAs transcribed from multiple genes. In Chapter 8, we discussed how backward slippage during DNA replication can lead to expansion of a microsatellite repeat. At least 14 different types of neurological diseases result from expansion of microsatellite regions within transcription units expressed in neurons. The resulting long regions of repeated simple sequences in nuclear pre-mRNAs of these neurons result in abnormalities in the relative concentrations of alternatively spliced mRNAs. For example, the most common of these types of diseases, myotonic dystrophy, results from increased copies of either CUG repeats in one transcript, in some patients, or CCUG repeats in another transcript, in other patients. When the number of these repeats increases to 10 or more times the normal number of repeats, abnormali ties are observed in the functions of two hnRNP proteins that bind to these repeated sequences. The abnormalities probably result because the hnRNPs are bound by the abnormally high concentrations of the repeats in the nuclei of neurons in these patients and cannot associate with other pre-mRNAs. This sequestration of the hnRNPs leads to alterations in the rate of splicing of different alternative splice sites in multiple pre-mRNAs that are normally regulated by these hnRNP proteins. Because of the importance of the proper regulation of alternative splicing for the normal function of neurons, multiple human neurological disorders are associated with abnormalities in the function of nuclear RNA-binding proteins and the expansion of microsatellite repeats that generate binding sites for splicing factors (Table 1).

Table1. Neurological Disorders with Links to Abnormalities in Alternative RNA Splicing

Expression of Dscam Isoforms in Drosophila Retinal Neurons

 The most extreme example of regulated alternative RNA processing yet uncovered occurs in expression of the Dscam gene in Drosophila. Mutations in this gene interfere with the normal synaptic connections made between retinal axons and dendrites during fly development. Analysis of the Dscam gene showed that it contains four groups of exons within which one of several possible exons is included in the final mature mRNA. The gene contains a total of 95 exons (Figure 4), generating 38,016 possible alter natively spliced isoforms! Drosophila mutants with a version of the gene that can be spliced in only about 22,000 different ways have specific defects in connectivity between neurons. These results indicate that expression of most of the possible Dscam isoforms through regulated RNA splicing helps to specify the tens of millions of different specific synaptic connections between neurons in the Drosophila brain. In other words, the correct wiring of neurons in the brain requires regulated RNA splicing.

Fig4. The Drosophila Dscam gene is processed into a vast number of alternative isoforms. Dscam encodes a cell-surface protein on neurons. The protein (bottom) is composed of ten different immunoglobulin (Ig) domains (ovals), six different fibronectin type III domains (rectangles), one transmembrane domain (yellow), and a C-terminal cytoplasmic domain (dark gray). The fully processed mRNA is shown as rectangles representing each exon, with the length of the rectangle corresponding to the length of the exons, and a green circle representing the 5’ cap. Each mRNA contains one of the 12 Ig2 exons shown in light blue (top), one of the 48 Ig3 exons shown in green, one of the 33 Ig7 exons shown in dark blue, and one of the 2 transmembrane exons shown in yellow. The exons shown in pink are spliced into each of the messages. Thus alternative splicing can generate 12 × 48 × 33 × 2 = 38,016 possible isoforms. See M. R. Sawaya et al., 2008, Cell 134:1007.

RNA Editing Alters the Sequences of Some Pre-mRNAs

In the mid-1980s, sequencing of numerous cDNA clones and corresponding genomic DNAs from multiple organisms led to the unexpected discovery of another type of pre-mRNA processing. In this type of processing, called RNA editing, the sequence of a pre-mRNA is altered; as a result, the sequence of a mature mRNA differs from that of the exons encoding it in genomic DNA.

RNA editing is widespread in the mitochondria of protozoans and plants as well as in chloroplasts. In the mitochondria of certain pathogenic trypanosomes, more than half the sequence of some mRNAs is altered from the sequence of the corresponding primary transcripts. Additions and deletions of specific numbers of Us follow templates provided by base-paired short “guide” RNAs. These RNAs are encoded by thousands of small circular DNA molecules concatenated to many fewer large DNA molecules. The reason for this baroque mechanism for encoding mitochondrial proteins in such protozoans is not clear. But this system does represent a potential target for drugs to inhibit the complex processing enzymes essential to the microbe that do not exist in the cells of its human or other vertebrate hosts.

In higher eukaryotes, RNA editing is much rarer, and thus far, only single-base changes have been observed. Such minor editing, however, turns out to have significant functional consequences in some cases. An important example of RNA editing in mammals involves the APOB gene, which encodes two alternative forms of a serum protein that is central to the uptake and transport of cholesterol. Consequently, it is important in the pathogenic processes that lead to atherosclerosis, the arterial disease that is the major cause of death in the developed world. The APOB gene encodes both the serum protein apolipoprotein B-100 (apoB-100), which is expressed in hepatocytes, the major cell type in the liver, and apoB-48, which is expressed in intestinal epithelial cells. The 240-kDa apoB-48 corresponds to the N-terminal region of the 500-kDa apoB-100. Both ApoB proteins are components of the large lipoprotein complexes we described in Chapter 7, which trans port lipids in the serum. However, only low-density lipoprotein (LDL) complexes, which contain apoB-100 on their surface, deliver cholesterol to body tissues by binding to the LDL receptor that is present on all cells.

The cell-type-specific expression of the two forms of ApoB results from editing of ApoB pre-mRNA so as to change the nucleotide at position 6666 in the sequence from a C to a U. This alteration, which occurs only in intestinal cells, converts a CAA codon for glutamine to a UAA stop codon, leading to synthesis of the shorter apoB-48 (Figure 5). Studies with the partially purified enzyme that performs the post-transcriptional deamination of C₆₆₆₆ to U shows that it can recognize and edit an RNA as short as 26 nucleotides containing the sequence surrounding C₆₆₆₆ in the ApoB primary transcript.

Fig5. RNA editing of APOB pre-mRNA. The APOB mRNA produced in the liver has the same sequence as the exons in the primary transcript. This mRNA is translated into apoB-100, which has two functional domains: an N-terminal domain (green) that associates with lipids and a C-terminal domain (orange) that binds to LDL receptors on cell membranes. In the APOB mRNA produced in the intestine, however, the CAA codon in exon 26 is edited to a UAA stop codon. As a result, intestinal cells produce apoB-48, which corresponds to the N-terminal domain of apoB-100. See P. Hodges and J. Scott, 1992, Trends Biochem. Sci. 17:77.

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مواضيع ذات صلة

Transcription Initiation by Pol I and Pol III Is Analogous to That by Pol II

The Mediator Complex Forms a Molecular Bridge Between Activation Domains and Pol II

Pioneer Transcription Factors Initiate the Process of Gene Activation During Cellular Differentiation

Genetic Material can be Altered & Rearranged

Translation and protein production

Epigenetic Regulation of Transcription

Chromatin-Remodeling Complexes Help Activate or Repress Transcription

Activators Can Direct Histone Acetylation at Specific Genes

Repressors Can Direct Histone Deacetylation at Specific Genes

Formation of Heterochromatin Silences Gene Expression at Telomeres, near Centromeres, and in Other Regions

Multiprotein Complexes Form on Enhancers

Transcription Factor Interactions Increase Gene-Control Options