Chapter 19: RNA Splicing and Processing Chapter Opener: © Laguna Design/Getty Images. CHAPTER OUTLINE 19.1 Introduction 19.2 The 5′ End of Eukaryotic mRNA Is Capped 19.3 Nuclear Splice Sites Are Short Sequences 19.4 Splice Sites Are Read in Pairs 19.5 Pre-mRNA Splicing Proceeds Through a Lariat 19.6 snRNAs Are Required for Splicing 19.7 Commitment of Pre-mRNA to the Splicing Pathway booksmedicos.org 19.8 The Spliceosome Assembly Pathway 19.9 An Alternative Spliceosome Uses Different snRNPs to Process the Minor Class of Introns 19.10 Pre-mRNA Splicing Likely Shares the Mechanism with Group II Autocatalytic Introns 19.11 Splicing Is Temporally and Functionally Coupled with Multiple Steps in Gene Expression 19.12 Alternative Splicing Is a Rule, Rather Than an Exception, in Multicellular Eukaryotes 19.13 Splicing Can Be Regulated by Exonic and Intronic Splicing Enhancers and Silencers 19.14 trans-Splicing Reactions Use Small RNAs 19.15 The 3′ Ends of mRNAs Are Generated by Cleavage and Polyadenylation 19.16 3′ mRNA End Processing Is Critical for Termination of Transcription 19.17 The 3′ End Formation of Histone mRNA Requires U7 snRNA 19.18 tRNA Splicing Involves Cutting and Rejoining in Separate Reactions 19.19 The Unfolded Protein Response Is Related to tRNA Splicing 19.20 Production of rRNA Requires Cleavage Events and Involves Small RNAs 19.1 Introduction booksmedicos.org RNA is a central player in gene expression. It was first characterized as an intermediate in protein synthesis, but since then many other RNAs that play structural or functional roles at various stages of gene expression have been discovered. The involvement of RNA in many functions involved with gene expression supports the general view that life may have evolved from an “RNA world” in which RNA was originally the active component in maintaining and expressing genetic information. Many of these functions were subsequently assisted or taken over by proteins, with a consequent increase in versatility and probably efficiency. All RNAs studied thus far are transcribed from their respective genes and (particularly in eukaryotes) require further processing to become mature and functional. Interrupted genes are found in all groups of eukaryotic organisms. They represent a small proportion of the genes of unicellular eukaryotes, but the majority of genes in multicellular eukaryotic genomes. Genes vary widely according to the numbers and lengths of introns, but a typical mammalian gene has seven to eight exons spread out over about 16 kb. The exons are relatively short (about 100 to 200 bp), and the introns are relatively long (almost 1 kb) (see the chapter titled The Interrupted Gene). The discrepancy between the interrupted organization of the gene and the uninterrupted organization of its mRNA requires processing of the primary transcription product. The primary transcript has the same organization as the gene and is called the pre-mRNA. Removal of the introns from pre-mRNA leaves an RNA molecule with an average length of about 2.2 kb. Removal of introns is a major part of the processing of RNAs in all eukaryotes. The process by which the introns are removed is called RNA splicing. Although interrupted genes are relatively rare in most unicellular/oligocellular eukaryotes (such as the yeast booksmedicos.org Saccharomyces cerevisiae), the overall proportion underestimates the importance of introns because most of the genes that are interrupted encode relatively abundant proteins. Splicing is therefore involved in the production of a greater proportion of total mRNA than would be apparent from analysis of the genome, perhaps as much as 50%. One of the first clues about the nature of the discrepancy in size between nuclear genes and their products in multicellular eukaryotes was provided by the properties of nuclear RNA. Its average size is much larger than mRNA, it is very unstable, and it has a much greater sequence complexity. Taking its name from its broad size distribution, it is called heterogeneous nuclear RNA (hnRNA). The physical form of hnRNA is a ribonucleoprotein particle, hnRNP, in which the hnRNA is bound by a set of abundant RNA-binding proteins. Some of the proteins may have a structural role in packaging the hnRNA; several are known to affect RNA processing or facilitate RNA export out of the nucleus. Splicing occurs in the nucleus, together with the other modifications that are made to newly synthesized RNAs. The process of expressing an interrupted gene is reviewed in FIGURE 19.1. The transcript is capped at the 5′ end, has the introns removed, and is polyadenylated at the 3′ end. The RNA is then transported through nuclear pores to the cytoplasm, where it is available to be translated. booksmedicos.org FIGURE 19.1 RNA is modified in the nucleus by additions to the 5′ and 3′ ends and by splicing to remove the introns. The splicing event requires breakage of the exon–intron junctions and joining of the ends of the exons. Mature mRNA is transported through nuclear pores to the cytoplasm, where it is translated. With regard to the various processing reactions that occur in the nucleus, we should like to know at what point splicing occurs vis-à- vis the other modifications of RNA. Does splicing occur at a particular location in the nucleus, and is it connected with other events—for example, transcription and/or nucleocytoplasmic transport? Does the lack of splicing make an important difference in the expression of uninterrupted genes? With regard to the splicing reaction itself, one of the main questions is how its specificity is controlled. What ensures that the ends of each intron are recognized in pairs so that the correct sequence is removed from the RNA? Are introns excised from a precursor in a particular order? Is the maturation of RNA used to regulate gene expression by discriminating among the available precursors or by changing the pattern of splicing? booksmedicos.org Besides RNA splicing to remove introns, many noncoding RNAs also require processing to mature, and they play roles in diverse aspects of gene expression. 19.2 The 5′ End of Eukaryotic mRNA Is Capped KEY CONCEPTS A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–5′ link. The capping process takes place during transcription and may be important for release from pausing of transcription. The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated. The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation. Transcription starts with a nucleoside triphosphate (usually a purine, A or G). The first nucleotide retains its 5′-triphosphate group and makes the usual phosphodiester bond from its 3′ position to the 5′ position of the next nucleotide. The initial sequence of the transcript can be represented as: A 5′ppp /GpNpNpNp … However, when the mature mRNA is treated in vitro with enzymes that should degrade it into individual nucleotides, the 5′ end does not give rise to the expected nucleoside triphosphate. Instead it contains two nucleotides that are connected by a 5′–5′ triphosphate linkage and also bear a methyl group. The terminal base is always booksmedicos.org a guanine that is added to the original RNA molecule after transcription. Addition of the 5′ terminal G is catalyzed by a nuclear enzyme, guanylyl-transferase (GT). In mammals, GT has two enzymatic activities, one functioning as the triphosphatase to remove the two phosphates in GTP and the other as the guanylyl-transferase to fuse the guanine to the original 5′-triphosphate terminus of the RNA. In yeast, these two activities are carried out by two separate enzymes. The new G residue added to the end of the RNA is in the reverse orientation from all the other nucleotides: 5′Gppp + 5′pppApNpNp … → Gppp5′–5′ApNpNp … + pp + p This structure is called a cap. It is a substrate for several methylation events. FIGURE 19.2 shows the full structure of a cap after all possible methyl groups have been added. The most important event is the addition of a single methyl group at the 7 position of the terminal guanine, which is carried out by guanine-7- methyltransferase (MT). booksmedicos.org FIGURE 19.2 The cap blocks the 5′ end of mRNA and can be methylated at several positions. Although the capping process can be accomplished in vitro using purified enzymes, the reaction normally takes place during transcription. Shortly after transcription initiation, Pol II is paused about 30 nucleotides downstream from the initiation site, waiting for the recruitment of the capping enzymes to add the cap to the 5′ end of nascent RNA. Without this protection, nascent RNA may be vulnerable to attack by 5′–3′ exonucleases, and such trimming may induce the Pol II complex to fall off of the DNA template. Thus, the process of capping is important for Pol II to enter the productive mode of elongation to transcribe the rest of the gene. In this regard, the pausing mechanism for 5′ capping represents a checkpoint for transcription reinitiation from the initial pausing site. booksmedicos.org In a population of eukaryotic mRNAs, every molecule contains only one methyl group in the terminal guanine, generally referred to as a monomethylated cap. In contrast, some other small noncoding RNAs, such as those involved in RNA splicing in the spliceosome (see the section later in this chapter titled snRNAs Are Required for Splicing), are further methylated to contain three methyl groups in the terminal guanine. This structure is called a trimethylated cap. The enzymes for these additional methyl transfers are present in the cytoplasm. This may ensure that only some specialized RNAs are further modified at their caps.