2. Nuclear splice junctions are interchangeable but are read in pairs

22.1 Introduction

Key terms defined in this section

RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA.

Interrupted genes are found in all classes of organisms. They represent a minor proportion of the genes of the very lowest eukaryotes, but the vast majority of genes in higher eukaryotic genomes. Genes vary widely according to the numbers and lengths of introns, but a typical mammalian gene has 7 V8 exons spread out over ~16 kb. The exons are relatively short (~100 V200 bp), and the introns are relatively long (>1 kb) (see 2 From genes to genomes).

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 sometimes called the pre-mRNA. Removal of the introns from pre-mRNA leaves a typical messenger of ~2.2 kb. The process by which the introns are removed is called RNA splicing.

One of the first clues about the nature of the discrepancy in size between nuclear genes and their products in higher 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 was called heterogeneous nuclear RNA (hnRNA). It includes pre-mRNA, but could also include other transcripts (that is, which are not ultimately processed to mRNA; for review see Lewin, 1975).

Figure 22.1 hnRNA exists as a ribonucleoprotein particle organized as a series of beads.

The physical form of hnRNA is a ribonucleoprotein particle (hnRNP), in which the hnRNA is bound by proteins. As characterized in vitro, an hnRNP particle takes the form of beads connected by a fiber. The structure is summarized in Figure 22.1. The most abundant proteins in the particle are the core proteins, but other proteins are present at lower stoichiometry, making a total of ~20 proteins. The proteins typically are present at ~10⁸ copies per nucleus, compared with ~10⁶ molecules of hnRNA. The exact structure of the hnRNP, and the functional implications of RNA’s packaging in this manner, remain to be determined (for review see Dreyfuss et al., 1993).

Figure 22.2 RNA is modified in the nucleus by additions to the 5 F and 3 F 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; the expanded illustration shows the principle schematically, but not the actual order of events. Mature mRNA is transported through nuclear pores to the cytoplasm, where it is translated.

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 22.2. The transcript is capped at the 5′ end (as we saw in 5 Messenger RNA), has the introns removed (as we see in this chapter), and is polyadenylated at the 3′ end (this chapter). The RNA is then transported through nuclear pores to the cytoplasm, where it is available to be 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, 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?

We can identify several types of splicing systems:

• Introns are removed from the nuclear RNAs of higher eukaryotes by a system that recognizes only short consensus sequences conserved at exon-intron boundaries and within the intron. This reaction requires a large splicing apparatus, which takes the form of an array of proteins and ribonucleoproteins that functions as a large particulate complex (the spliceosome).
  
• Excision of certain introns is an autonomous property of the RNA itself. Two groups of introns with this capacity are found in diverse locations. Each forms a characteristic type of secondary/tertiary structure. The sequences of one group are related to those of nuclear introns. The ability of RNA to show enzymatic activities is seen also in the self-cleavage of viroid RNAs and the catalytic activity of RNAase P. We discuss these reactions in 23 Catalytic RNA.
  
• The removal of introns from yeast nuclear tRNA precursors involves enzymatic activities whose dealings with the substrate resemble those of the tRNA processing enzymes, in which a critical feature is the conformation of the tRNA precursor.

Two fundamental types of mechanism are employed in splicing. All introns except those of nuclear pre-tRNAs are excised by transesterification reactions, although the catalytic and other components that are involved are distinct in each case. The introns of nuclear pre-tRNA are removed by cleavage and ligation.

Many of the introns with autonomous capacity to splice are mobile, that is, they have the ability to insert copies at new locations. This implies that they are likely to have originated by insertion into pre-existing genes (see 23 Catalytic RNA). It remains speculative whether introns of higher eukaryotic nuclear genes originated by insertion or were part of the original construction of the gene. It seems inevitable that introns of nuclear pre-tRNA genes must have originated by insertion into pre-existing genes.

Reviews

Dreyfuss, G. et al. (1993). hnRNP proteins and the biogenesis of mRNA. Ann. Rev. Biochem 62, 289-321.

Lewin, B. (1975). Units of transcription and translation: sequence components of hnRNA and mRNA. Cell 4, 77-93.