Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders

Abstract

The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.

FIGURE 1.

Patterns of TSL addition to Trichinella spiralis mRNAs. (A) Graph showing the number of times that each TSL was detected at splice acceptor sites, identified via 5′RACE (gray bars) or in a survey of publicly available EST sequences (∼22,000) (black bars). (B–D) Instances of TSL trans-splicing detected in 5′RACE analysis of blgs-1 and genes represented by BG731798 (RNA helicase) and BQ732141 (bcl-X_L). (B) An example of a gene that utilizes a classic outron splice acceptor site: a splice site that is involved in spliced leader trans-splicing, but not in cis-splicing. (C,D) Examples of trans-splice acceptor sites that can also participate in cis-splicing to upstream donor splice sites. Exons are denoted by boxes, with shading used to indicate open reading frames; unshaded regions denote untranslated regions. Introns and intergenic regions are represented by horizontal lines. The spliced leader is represented by a black box. In each case the number of representative clones as a fraction of the total number analyzed is given. The scale bar indicates 100 nt.

FIGURE 2.

Identification and characterization of spliced leader RNA genes and transcripts. (A) Schematic showing the distribution of the 19 SL RNA genes identified on supercontig 0. Each SL RNA gene is represented by a numbered arrow head that indicates the direction in which it is transcribed, the number indicating the tslr gene number. The tslr genes that encode identical TSLs are indicated by numbered suffixes. The position of the 5S RNA gene cluster is shown by diverging gray arrowheads. The coordinates show the position of the region along supercontig 0, given in base pairs. (B) Predicted secondary structures for SL RNAs derived from tslr-1 and tslr-14. The spliced leader sequences are depicted in white with dark countershading. Arrows indicate the spliced leader-intron boundary. (C) Northern blot of muscle larvae RNA probed with TSL-1 and TSL-14 radiolabeled oligonucleotides. Arrow indicates expected size of the two SL RNAs.

FIGURE 3.

Nematode phylogeny showing two possible scenarios to explain extant spliced leader complements. The phylogeny shown is based upon SSU rDNA-based studies (Aleshin et al. 1998; Blaxter et al. 1998; Holterman et al. 2006). All recent studies are agreed on the relative position of C. elegans with respect to T. spiralis, though the exact topologies differ. Boxes indicate SL RNA genes, with the shading used to indicate distinct genes. In model 1, the ancestor of all extant nematodes possessed multiple, different SL RNA genes, which were maintained in the lineage leading to T. spiralis. In this model, all but one of these were lost in Rhabditida lineage, the surviving one becoming the ancestor of the SL1 and SL2 RNA genes. In model 2, the ancestral nematode possessed only one (possibly multicopy) SL1-like gene, that in the Dorylaimia lineage underwent multiple rounds of duplication and diversification, but was maintained in the Rhabditida lineage. Information on trans-splicing in Enoplid nematodes should shed light on which of these models is correct.