Spliced leader trans-splicing

Abstract

What is spliced leader (SL) trans-splicing? It is an mRNA maturation process, similar to intron splicing, which has been shown to occur in a limited number of eukaryotes. In SL trans-splicing, the cell replaces nucleotides at the 5′ end of some pre-mRNAs with those of a special class of small nuclear RNAs, called SL RNAs. These are short molecules with two functionally distinct halves: the 5′ half consists of the leader sequence that is transferred to a pre-mRNA, along with the SL RNA's methylguanosine cap; the 3′ half contains a binding site for the Sm protein complex, which binds many of the RNAs involved in intron splicing. These two halves are separated by a splice donor site, a GT dinucleotide. Nuclear machinery trans-splices the leader sequence to splice acceptor sites (AG dinucleotides) in the 5′ region of target pre-mRNAs. As a result, many mRNAs in SL trans-splicing species have a common sequence at the 5′ end.How is SL trans-splicing related to intron splicing? The mechanism of SL trans-splicing is very similar to cis- (intron) splicing (Figure 1Figure 1). In both cases, the 2′ hydroxyl group of a nucleotide (usually adenosine) severs the pre-mRNA backbone at the splice donor site, freeing the upstream exon to displace the intron sequence at the splice acceptor. In intron splicing, the splice donor and acceptor sites lie on the same strand of RNA, separated by the intron sequence, which contains the branch point adenosine. For SL trans-splicing, the splice donor site of the SL RNA is attacked by an adenosine between the 5′ end of the pre-mRNA and its SL addition site. The region of the pre-mRNA upstream of the SL addition site, which is removed when the leader sequence is attached, is known as the ‘outron’ — it is ‘outside’ the gene, whereas introns are ‘inside’.Figure 1The leader sequence (yellow box) of an SL RNA is attached to the first exon (orange box) of a pre-mRNA by a trans-splicing reaction.The outron of the pre-mRNA and the intron-like portion of the SL RNA form a ‘Y’ branched byproduct, similar to the lariat structure formed during intron splicing. The 2,2,7-trimethylguanosine cap structure (or ‘Cap 4’ in trypanosomes) found on SL RNAs and trans-spliced mRNAs (blue circles) differs from the 7-methylguanosine caps typically found on mRNAs (red circles). These caps impart novel properties to trans-spliced messages.View Large Image | View Hi-Res Image | Download PowerPoint SlideStrikingly, experiments in the nematode Caenorhabditis elegans demonstrated that the relative location of the donor and acceptor sites is sufficient to determine whether a splice acceptor is cis- or trans-spliced. Cis-spliced acceptors can be converted to trans-splicing acceptors if the donor site upstream is mutated, and similarly an SL addition site will be cis-spliced if a donor site is inserted upstream.What is the function of SL trans-splicing? SL addition provides the cell with an alternative way of capping mRNAs, a modification required for mRNA stability, transport and translation. The standard capping machinery is typically recruited by RNA polymerase II at the beginning of transcription to cap the growing RNA. In trypanosomes, SL addition is used to cap a subset of pre-mRNAs transcribed by RNA polymerase I, which does not recruit the capping machinery. In a wider range of eukaryotes, SL addition allows the formation of operons — adjacent genes that are transcribed as a single primary transcript. The cleavage reaction that precedes polyadenylation of each gene effectively chops the transcript into smaller pieces. Since only the message from the 5′ end retains the transcript's original cap, SL trans-splicing is needed to cap the remaining fragments.Addition of an SL has also been shown to affect the translation rate of some genes, add missing start codons and trim off outron sequences. But while the role of SL addition in these processes is well established, the benefit of transcription by polymerase I, inclusion in an operon, translational regulation, substitution of a start codon and outron removal is not immediately obvious for most trans-spliced genes.How is it phylogenetically distributed? The complete phylogenetic distribution of SL trans-splicing is not currently known. To date, SL trans-splicing has been found in six diverse groups of eukaryotes: nematodes, flatworms, cnidarians, ascidians, rotifers and euglenozoans. But it has not been detected in other well-studied eukaryotic taxa, such as fungi, plants, vertebrates and arthropods.How did it evolve? There are two competing hypotheses describing the origin of SL trans-splicing. The ‘SL trans-splicing early’ hypothesis proposes that SL trans-splicing was present in the ancestral eukaryote and subsequently lost in most phyla. This hypothesis is supported in part by the continuing discovery of SL trans-splicing in an expanding range of eukaryotes. The ‘SL trans-splicing late’ hypothesis proposes that the process has emerged several times independently, and that features unique to SL addition — the SL RNA, trans-spliceosome-specific proteins, SL-specific translation enhancer proteins, operons, and so on — are also independently derived in these lineages. This hypothesis is supported by the observation that the few unique components shown to be involved in SL addition are not obviously conserved across different trans-splicing phyla. Further studies into the mechanism and effects of SL trans-splicing in different phyla will help to clarify its origin.Why should I care? Many of the organisms that perform SL trans-splicing, such as trypanosomes, flatworms and nematodes are pathogenic to humans. Drugs that target components of the trans-spliceosome or disrupt the downstream effects of SL trans-splicing may be highly effective against these parasitic species, while causing little harm to patients.