Regulation of PremRNA Processing

Now that we've seen how pre-mRNAs are processed into mature, functional mRNAs, we consider how regulation of this process can contribute to gene control. Recall from Chapter 10 that higher eukaryotes contain both simple and complex transcription units. 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 can be processed in alternative ways to yield different mRNAs that encode distinct proteins (see Figure 10-2).

Alternative Splicing Is the Primary Mechanism for Regulating mRNA Processing

The results from recent genomic studies suggest that the number of complex transcription units may be much greater than previously realized. Researchers estimate that up to =60 percent of all transcription units in the human genome are complex. This estimate comes from comparison of the genomic DNA sequence with expressed sequence tags (ESTs), which are randomly sequenced segments of cDNA (Chapter 9). Comparison of these cDNA sequences with genomic DNA sequence shows the locations of exons and introns in genomic DNA. The finding of alternative combinations of exons expressed from a region of genomic DNA reveals the existence of a complex transcription unit. Even though this estimate may be elevated somewhat by inclusion in the EST database of rare RNAs that result from mistakes in RNA processing, it is clear that complex transcription units account for a large portion of the protein-coding sequences in humans. This finding and the discovery of multiple examples of regulated RNA processing indicate that alternative RNA processing of complex pre-mRNAs produced from such transcription units is a significant gene-control mechanism in higher eukaryotes.

Although 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. Such alternative processing pathways usually are regulated, often in a cell-type-specific manner; that is, one possible mRNA is expressed in one type of cell or tissue, while another is expressed in different cells or tissues. In Chapter 4, 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, which is spliced differently in the two cell types to yield two different mRNAs (see Figure 4-15). In other cases, alternative processing may occur in the same cell type in response to different developmental or environmental signals. First we discuss one of the best-understood examples of regulated RNA processing and then consider its consequences in 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. 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.

The Sxl protein, encoded by the sex-lethal gene, is the first protein to act in the cascade. Early in development, this 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. However, in the absence of early Sxl protein, the sex-lethal pre-mRNA in male embryos is spliced 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.

In contrast, the Sxl protein expressed in early female embryos directs splicing of the sex-lethal pre-mRNA so that a functional sex-lethal mRNA is produced (Figure 12-14a). 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. As a consequence, the U1 snRNP bound to the 3' end of exon 2 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 splicing of exon 2 to 4 and skipping of exon 3. The resulting female-specific sex-lethal mRNA is translated into functional Sxl protein, which reinforces 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 that prevents translation of functional Sxl protein.

Sxl protein also regulates alternative RNA splicing of the transformer gene pre-mRNA (Figure 12-14b). In male embryos, where no Sxl is expressed, exon 1 is spliced to exon 2, which contains a stop codon that prevents synthesis of a functional protein. In female embryos, however, binding of Sxl protein to 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 RRM domains in the protein (see Figure 12-3). 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, eliminating exon 2 with its stop codon. The resulting female-specific transformer mRNA, which contains additional constitutively spliced exons, is translated into functional Transformer (Tra) protein.

Finally, Tra protein regulates the alternative processing of pre-mRNA transcribed from the double-sex gene (Figure 12-14c). In female embryos, a complex of Tra and two constitutively expressed proteins, Rbp1 and Tra2, directs splicing of exon 3 to exon 4 and also promotes cleavage/

Pre-mRNAs mRNAs

Pre-mRNAs mRNAs

▲ FIGURE 12-14 Cascade of regulated splicing that controls sex determination via expression of sex-lethal (sxl), transformer (tra), and double-sex (dsx) genes 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. [Adapted from M. J. Moore et al., 1993, in R. Gesteland and J. Atkins, eds., The RNA World, Cold Spring Harbor Press, pp. 303-357]

polyadenylation at the alternative poly(A) site at the 3' end of exon 4. 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. As a result of the cascade of regulated RNA processing depicted in Figure 12-14, different Dsx proteins are expressed in male and female embryos. The male Dsx protein is a transcriptional repressor that inhibits the expression of genes required for female development. Conversely, the female Dsx protein represses transcription of genes required for male development.

Figure 12-15 illustrates how the Tra/Tra2/Rbp1 complex is thought to interact with double-sex pre-mRNA. Recent studies have shown that Rbp1 and Tra2 are SR proteins, similar to those discussed previously. They mediate the cooperative binding of the Tra/Tra2/Rbp1 complex to six exonic splicing enhancers in exon 4. The bound Tra2 and Rbp1 proteins then promote the binding of U2AF and U2 snRNP to the 3' end of the intron between exons 3 and 4, just as other SR proteins do for constitutively spliced exons (see Figure 12-11). The Tra/Tra2/Rbp1 complexes may also enhance binding of the cleavage/polyadnylation complex to the 3' end of exon 4.

► FIGURE 12-15 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 binding of Tra/Tra2/Rbp1 complexes to six sites 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. [Adapted from T Maniatis and B. Tasic, 2002, Nature 418:236.]

Splicing Repressors and Activators Control Splicing at Alternative Sites

As is evident from Figure 12-14, 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 (see Figure 4-15). Alternatively, a Tra-like splicing activator expressed in fibroblasts might activate the splice sites associated with the fibronectin EIIIA and EIIIB exons, leading to inclusion of these exons in the mature mRNA. Experimental examination in some systems has revealed that inclusion of an exon in some cell types versus skipping of the same exon in other cell types results from the combined influence of several splicing repressors and enhancers.

Alternative splicing of exons is especially common in the nervous system, generating multiple isoforms of many proteins required for neuronal development and function in both vertebrates and invertebrates. The primary transcripts from these genes often show quite complex splicing patterns that can generate several different mRNAs, with different spliced forms expressed in different anatomical locations within the central nervous system. We consider two remarkable examples that illustrate the critical role of this process in neural function.

Expression of Slo Isoforms 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 frequency («50 Hz) are found at one end of the tubular cochlea that makes up the inner ear; cells responding to high frequency («5000 Hz) are found at the other end (Figure 12-16a). Cells in between 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.

The gene encoding this channel (called slo, after the homologous Drosophila gene) is expressed as multiple, alternatively spliced mRNAs. The various Slo proteins encoded by these alternative mRNAs open at different Ca2+ concentrations. Hair cells with different response frequencies express different isoforms of the Slo 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 alternative exons are utilized, permitting the expression of 576 possible isoforms (Figure 12-16b). PCR analysis of slo mRNAs from individual hair cells has shown that each hair cell expresses a mixture of different alternative slo mRNAs, with different forms predominating in different cells according to their position along the cochlea. This remarkable arrangement suggests that splicing of the slo pre-mRNA is regulated in response to extracellular signals that inform the cell of its position along the cochlea.

Recent results have shown that splicing at one of the alternative splice sites in the slo pre-mRNA in the rat is suppressed when a specific protein kinase is activated by depolarization of the neuron in response to synaptic activity from interacting neurons. This observation raises the possibility that a splicing repressor specific for this site may be activated when it is phosphorylated by this protein kinase,

▲ FIGURE 12-16 Role of alternative splicing of slo mRNA in the perception of sounds of different frequency. (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 Slo protein contains seven transmembrane a helices (S0-S6), which associate to form the K+ channel. The cytosolic domain, which includes four hydrophobic regions (S7-S10), regulates opening of the channel in response to Ca2+. Isoforms of the Slo 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 Slo isoforms. [Adapted from K. P Rosenblatt et al., 1997, Neuron 19:1061.]

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.

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 connections made by the axons of retinal neurons with neurons in a specific region of the brain during fly development. Analysis of the Dscam gene shows that it contains 95 alternatively spliced exons that could be spliced to generate over 38,000 possible isoforms! These results raise the possibility that the expression of different Dscam isoforms through regulated RNA splicing helps to specify the tens of thousands of different specific synaptic connections made between retinal and brain neurons. In other words, the correct wiring of neurons in the brain may depend on regulated RNA splicing.

RNA Editing Alters the Sequences of Pre-mRNAs

Sequencing of numerous cDNA clones and of the corresponding genomic DNAs from multiple organisms led in the mid-1980s to the unexpected discovery of a previously unrecognized 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 the corresponding mature mRNA differs from the exons encoding it in genomic DNA.

RNA editing is widespread in the mitochondria of protozoans and plants and also in chloroplasts; in these organelles, more than half the sequence of some mRNAs is altered from the sequence of the corresponding primary tran scripts. 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 important functional consequences in some cases.

An important example of RNA editing in mammals involves the apoB gene, which encodes two alternative forms of the serum protein apolipoprotein B (apoB): apoB-100 expressed in hepatocytes and apoB-48 expressed in intestinal epithelial cells. The «240-kDa apoB-48 corresponds to the N-terminal region of the «500-kDa apoB-100. As we detail in Chapter 18, both apoB proteins are components of large lipoprotein complexes that transport 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 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 12-17). 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 with the sequence surrounding C6666 in the apoB primary transcript.

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