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▲ FIGURE 12-10 Schematic diagram of RNA polymerase II with the CTD extended. The length of the fully extended yeast RNA polymerase II carboxyl-terminal domain (CTD) and the linker region that connects it to the polymerase is shown relative to the globular domain of the polymerase. The CTD of mammalian

▲ FIGURE 12-10 Schematic diagram of RNA polymerase II with the CTD extended. The length of the fully extended yeast RNA polymerase II carboxyl-terminal domain (CTD) and the linker region that connects it to the polymerase is shown relative to the globular domain of the polymerase. The CTD of mammalian

RNA polymerase II is twice as long. In its extended form, the CTD can associate with multiple RNA-processing factors simultaneously. [From P Cramer, D. A. Bushnell, and R. D. Kornberg, 2001, Science 292:1863.]

splicing together separate RNA molecules. This process, referred to as trans-splicing, is also used in the synthesis of 10-15 percent of the mRNAs in the nematode (round worm) Caenorhabditis elegans, an important model organism for studying embryonic development. Trans-splicing is carried out by snRNPs by a process similar to the splicing of exons in a single pre-mRNA.

Chain Elongation by RNA Polymerase II Is Coupled to the Presence of RNA-Processing Factors

The carboxyl-terminal domain (CTD) of RNA polymerase II is composed of multiple repeats of a seven-residue (heptapeptide) sequence. When fully extended, the CTD domain in the yeast enzyme is about 65 nm long (Figure 12-10); the CTD in human polymerase is about twice as long. The remarkable length of the CTD apparently allows multiple proteins to associate simultaneously with a single RNA polymerase II molecule. For instance, as mentioned earlier, the enzyme that adds the 5' cap to nascent transcripts associates with the phosphorylated CTD shortly after transcription initiation. In addition, RNA splicing and polyadenylation factors have been found to associate with the phosphorylated CTD. As a consequence, these processing factors are present at high local concentrations when splice sites and poly(A) signals are transcribed by the polymerase, enhancing the rate and specificity of RNA processing.

Recent results indicate that the association of RNA splicing factors with the phosphorylated CTD also stimulates transcription elongation. Thus chain elongation is coupled to the binding of RNA-processing factors to the phosphorylated CTD. This mechanism may ensure that a pre-mRNA is not synthesized unless the machinery for processing it is properly positioned.

SR Proteins Contribute to Exon Definition in Long Pre-mRNAs

The average length of an exon in the human genome is «150 bases, whereas the average length of an intron is much longer («3500 bases). The longest introns contain upwards of 500 kb! Because the sequences of 5' and 3' splice sites and branch points are so degenerate, multiple copies are likely to occur randomly in long introns. Consequently, additional sequence information is required to define the exons that should be spliced together in higher organisms with long introns.

The more sophisticated mechanism of exon recognition required in higher organisms entails a family of RNA-binding proteins, the ,SR proteins, that interact with sequences within exons called exonic splicing enhancers. SR proteins are characterized by having one or more RRM RNA-binding domains and several protein-protein interaction domains rich in serine and arginine residues. When bound to exonic splicing enhancers, SR proteins mediate the cooperative binding of U1 snRNP to a true 5' splice site and U2 snRNP to a branch point through a network of proteinprotein interactions that span across an exon (Figure 12-11). The complex of SR proteins, snRNPs, and other splicing factors (e.g., U2AF) that assemble across an exon, which has been called a cross-exon recognition complex, permits precise specification of exons in long pre-mRNAs.

In the transcription units of higher organisms with I long introns, exons not only encode the amino acid h'lUiHHii'H sequences of different portions of a protein but also contain binding sites for SR proteins. Mutations that interfere with the binding of an SR protein to an exonic splicing enhancer, even if they do not change the encoded amino acid sequence, would prevent formation of the cross-exon recognition complex. As a result, the affected exon is "skipped" during splicing and not included in the final processed mRNA. The truncated mRNA produced in this case is either degraded or translated into a mutant, abnormally functioning protein. Recent studies have implicated this type of mutation in human genetic diseases. For example, spinal muscle atrophy is one of the most common genetic causes of childhood mortality. This disease results from mutations in a region of the genome containing two closely related genes, SMN1 and ,SMN2, that arose by gene duplication. ,SMN2 encodes a protein identical with SMN1, but it is expressed at much lower level because a silent mutation in one exon interferes with the binding of an SR protein, leading to exon skipping in most of the ,SMN2 mRNAs. The homologous SMNgene in the mouse, where there is only a single copy, is essential for cell viability. Spinal muscle atrophy in humans results from homozygous mutations that inactivate SMN1. The low level of protein translated from the small

▲ FIGURE 12-11 Exon recognition through cooperative binding of SR proteins and splicing factors to pre-mRNA.

The correct 5' GU and 3' AG splice sites are recognized by splicing factors on the basis of their proximity to exons. The exons contain exonic splicing enhancers (ESEs) that are binding sites for SR proteins. When bound to ESEs, the SR proteins interact with one another and promote the cooperative binding of the U1 snRNP to the 5' splice site of the downstream intron, the U2 snRNP to the branch point of the upstream intron, the 65- and 35-kD subunits of U2AF to the pyrimidine-rich region and AG 3' splice site of the upstream intron, and other splicing factors (not shown). The resulting RNA-protein cross-exon fraction of SMN2 mRNAs that are correctly spliced is sufficient to maintain cell viability during embryogenesis and fetal development, but it is not sufficient to maintain viability of spinal cord motor neurons in childhood, resulting in their death and the associated disease.

Approximately 15 percent of the single-base-pair mutations that cause human genetic diseases interfere with proper exon definition. Some of these mutations occur in 5' or 3' splice sites, often resulting in the use of nearby alternative "cryptic" splice sites present in the normal gene sequence. In the absence of the normal splice site, the cross-exon recognition complex recognizes these alternative sites. Other mutations that cause abnormal splicing result in a new consensus splice site sequence that becomes recognized in place of the normal splice site. Finally, some mutations can interfere with the binding of specific hnRNP proteins to pre-mRNAs that enhance or repress splicing at normal splice sites, as in the case of the .SMN2 gene. I

Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs

Under certain nonphysiological in vitro conditions, pure preparations of some RNA transcripts slowly splice out in-trons in the absence of any protein. This observation led to recognition that some introns are self-splicing. Two types of self-splicing introns have been discovered: group I introns, present in nuclear rRNA genes of protozoans, and group II introns, present in protein-coding genes and some rRNA and tRNA genes in mitochondria and chloroplasts of plants and fungi. Discovery of the catalytic activity of self-splicing introns revolutionized concepts about the functions of RNA. As discussed in Chapter 4, RNA is now thought to catalyze recognition complex spans an exon and activates the correct splice sites for RNA splicing. Note that the U1 and U2 snRNPs in this unit are not part of the same spliceosome. The U2 snRNP on the right forms a spliceosome with the U1 snRNP bound to the 5'-end of the same intron. The U1 snRNP shown on the right forms a spliceosome with the U2 snRNP bound to the branch point of the downstream intron (not shown), and the U2 snRNP on the left forms a spliceosome with a U1 snRNP bound to the 5' splice site of the upstream intron (not shown). Double-headed arrows indicate protein-protein interactions. [Adapted from T Maniatis, 2002, Nature 418:236; see also S. M. Berget, 1995, J. Biol. Chem. 270:2411.]

peptide-bond formation during protein synthesis in ribo-somes. Here we discuss the probable role of group II introns, now found only in mitochondrial and chloroplast DNA, in the evolution of snRNAs; the functioning of group I introns is considered in the later section on rRNA processing.

Even though their precise sequences are not highly conserved, all group II introns fold into a conserved, complex secondary structure containing numerous stem-loops (Figure 12-12a). Self-splicing by a group II intron occurs via two transesterification reactions, involving intermediates and products analogous to those found in nuclear pre-mRNA splicing. The mechanistic similarities between group II intron self-splicing and spliceosomal splicing led to the hypothesis that snRNAs function analogously to the stem-loops in the secondary structure of group II introns. According to this hypothesis, snRNAs interact with 5' and 3' splice sites of pre-mRNAs and with each other to produce a three-dimensional RNA structure functionally analogous to that of group II self-splicing introns (Figure 12-12b).

An extension of this hypothesis is that introns in ancient pre-mRNAs evolved from group II self-splicing introns through the progressive loss of internal RNA structures, which concurrently evolved into trans-acting snRNAs that perform the same functions. Support for this type of evolutionary model comes from experiments with group II intron mutants in which domain V and part of domain I are deleted. RNA transcripts containing such mutant introns are defective in self-splicing, but when RNA molecules equivalent to the deleted regions are added to the in vitro reaction, self-splicing occurs. This finding demonstrates that these domains in group II introns can be trans-acting, like snRNAs.

The similarity in the mechanisms of group II intron self-splicing and spliceosomal splicing of pre-mRNAs also

(a) Group II intron

(b) U snRNAs in spliceosome

(a) Group II intron

(b) U snRNAs in spliceosome

Pre-mRNA

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