Genes VII

In both mechanistic and evolutionary terms, splicing has been viewed as an intramolecular reaction, amounting essentially to a controlled deletion of the intron sequences at the level of RNA. In genetic terms, splicing occurs only in cis. This means that only sequences on the same molecule of RNA can be spliced together. The upper part of Figure 22.21 shows the normal situation. The introns can be removed from each RNA molecule, allowing the exons of that RNA molecule to be spliced together, but there is no intermolecular splicing of exons between different RNA molecules. We cannot say that trans splicing never occurs between pre-mRNA transcripts of the same gene, but we know that it must be exceedingly rare, because if it were prevalent the exons of a gene would be able to complement one another genetically instead of belonging to a single complementation group.

Some manipulations can generate trans-splicing. In the example illustrated in the lower part of Figure 22.21, complementary sequences were introduced into the introns of two RNAs. Base pairing between the complements should create an H-shaped molecule. This molecule could be spliced in cis, to connect exons that are covalently connected by an intron, or it could be spliced in trans, to connect exons of the juxtaposed RNA molecules. Both reactions occur in vitro.

Another situation in which trans-splicing is possible in vitro occurs when substrate RNAs are provided in the form of one containing a 5′ splice site and the other containing a 3′ splice site together with appropriate downstream sequences (which may be either the next 5′ splice site or a splicing enhancer). In effect, this mimics the splicing reaction that is used for long introns and short exons (see the right side of Figure 22.11), and shows that in vitro it is not necessary for the left and right splice sites to be on the same RNA molecule.

These results show that there is no mechanistic impediment to trans-splicing. They exclude models for splicing that require processive movement of a spliceosome along the RNA. It must be possible for a spliceosome to recognize the 5′ and 3′ splice sites of different RNAs when they are in close proximity.

Although trans-splicing is rare, it occurs in vivo in some special situations. One is revealed by the presence of a common 35 base leader sequence at the end of numerous mRNAs in the trypanosome. But the leader sequence is not coded upstream of the individual transcription units. Instead it is transcribed into an independent RNA, carrying additional sequences at its 3′ end, from a repetitive unit located elsewhere in the genome. Figure 22.22 shows that this RNA carries the 35 base leader sequence followed by a 5′ splice site sequence. The sequences coding for the mRNAs carry a 3′ splice site just preceding the sequence found in the mature mRNA (Sutton and Boothroyd, 1986).

If the leader and the mRNA are connected by a trans-splicing reaction, the 3′ region of the leader RNA and the 5′ region of the mRNA will in effect comprise the 5′ and 3′ halves of an intron. If splicing occurs by the usual nuclear mechanism, a 5′ V2′ link should form by a reaction between the GU of the 5′ intron and a branch sequence near the AG of the 3′ intron. Because the two parts of the intron are not covalently linked, this generates a Y-shaped molecule instead of a lariat (Murphy et al., 1986).

A similar situation is presented by the expression of actin genes in C. elegans. Three actin mRNAs (and some other RNAs) share the same 22 base leader sequence at the 5′ terminus. The leader sequence is not coded in the actin gene, but is transcribed independently as part of a 100 base RNA coded by a gene elsewhere. trans-splicing also occurs in chloroplasts (Krause and Hirsh, 1987).

The RNA that donates the 5′ exon for trans splicing is called the SL RNA (spliced leader RNA). The SL RNAs found in several species of trypanosomes and also in the nematode (C. elegans) have some common features. They fold into a common secondary structure that has three stem-loops and a single-stranded region that resembles the Sm-binding site. The SL RNAs therefore exist as snRNPs that count as members of the Sm snRNP class. Trypanosomes possess the U2, U4, and U6 snRNAs, but do not have U1 or U5 snRNAs. The absence of U1 snRNA can be explained by the properties of the SL RNA, which can carry out the functions that U1 snRNA usually performs at the 5′ splice site; thus SL RNA in effect consists of an snRNA sequence possessing U1 function, linked to the exon-intron site that it recognizes.

There are two types of SL RNA in C. elegans. SL1 RNA (the first to be discovered) is used for splicing to coding sequences that are preceded only by 5′ nontranslated region (the most common situation). SL2 RNA is used in cases in which a pre-mRNA contains two coding sequences; it is spliced to the second sequence, thus releasing it from the first, and allowing it to be used as an mRNA (Huang and Hirsh, 1989; Hannon et al., 1990; for review see Nilsen, 1993).

The trans-splicing reaction of the SL RNA may represent a step towards the evolution of the full nuclear splicing apparatus. The SL RNA provides in cis the ability to recognize the 5′ splice site, and this probably depends upon the specific conformation of the RNA. The remaining functions required for splicing are provided by independent snRNPs. The SL RNA can function without participation of proteins like those in U1 snRNP, which suggests that the recognition of the 5′ splice site depends directly on RNA.