Operon conservation and the evolution of trans-splicing in the phylum Nematoda

Abstract

The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

Figure 1. The Evolution of Operons and SL Usage through the Phylum Nematoda

Operons and SL usage in trans-splicing have been mapped onto a phylogeny of the Nematoda illustrating the relationships of the nematodes studied based on analysis of the small subunit rRNA (adapted from [37,38]). Conserved operons have been identified in Rhabditina, Tylenchina, and Spriurina (clades V, IV, and III of [37]). While SL1 may be a synapomorphy for the phylum, SL2-like SLs are apparently restricted to the Rhabditina, as is their use in trans-splicing to downstream genes in operons. An independent radiation of SL1-like SLs is used for downstream gene trans-splicing in the Tylenchina, while the Spirurina use canonical SL1.

Figure 2. Conservation of Operonic Structures in Distantly Related Nematodes

The genomic structure of four conserved nematode operons is shown. Exons are depicted with rectangles, and introns with thin lines. The gene structures and scales (in base pairs) are from the C. elegans genomic sequence; the structure of the operons and the exon/intron boundaries are conserved in C. briggsae. Arrows indicate direction of transcription, and the dotted lines linking arrows indicate operonic structures. The position of novel introns in other nematodes is indicated by open arrowheads. Where only a fragment of the operon has been isolated, the extents of the isolated fragment are indicated on the base scale by vertical dashed lines and the taxa thus affected by letters. The lollipop symbols indicate absence of the intron in the indicated species. (A) CEOP1032 containing rpl-27a and rpa-1: orthologues identified in A. suum, B. malayi, N. brasiliensis, O. tipulae, P. pacificus, and S. ratti. (B) CEOP1624 containing rpa-1 and tct-1: orthologues identified in A. suum, B. malayi, P. pacificus, S. ratti, and N. brasiliensis. (C) CEOP5428 containing fib-1 and rps-16: orthologues found in P. pacificus. (D) CEOP3416 containing rpl-36 and F37C12.3 (an acyl carrier protein): orthologues found in B. malayi, P. pacificus, and S. ratti. The genomic structure surrounding CEOP3416 is also conserved. It contains four genes (F37C12.1, .2, .3, and .4) and spans a gene on the opposite strand (F37C12.14, in an intron of F37C12.1). rps-14 is found immediately upstream, and rps-21 one gene downstream, on the opposite strand. The filled triangle indicates the presence in B. malayi and S. ratti of an additional gene that shows similarity to C. elegans F37C12.2. Ce-F37C12.2 is found downstream of F37C12.3 in the same operonic structure CEOP3416 in C. elegans and C. briggsae. The new intron annotated with an asterisk (*) is found in B. malayi and S. ratti, but the two introns are separated by nine nucleotides of coding sequence in a protein-driven alignment: the orthology of these introns is thus debatable.

Figure 3. Mapping the 5′ End of Bm-rpa-1 mRNA and Isolation of Processing Intermediates of the Bm-rpl-27a/rpa-1 Polycistronic pre-mRNA

(A) An autoradiograph showing the primer extension products from B. malayi rpa-1 mRNAs. The single observed product is 248 bp. This is consistent with the expected size of an SL1 trans-spliced cDNA. L, M13 sequencing ladder; S, primer extension product. (B) The processing intermediates of the rpl-27a and rpa-1 polycistron amplified by RT-PCR. Fragment 1: no processing, introns in both genes present. Fragment 2: processing intermediate with rpl-27a intron removed. Fragment 3: processing intermediate with both the rpl-27a and the rpa-1 introns removed. +, reaction with reverse transcriptase added; −, sham reaction with no reverse transcriptase added; M, DNA size markers.

Figure 4. The Evolution of Nematode SLs

(A) Consensus maximum parsimony phylogram of SL relationships. (B) Majority-rule cladogram indicating percentage representation of nodes in 10,000 trees of the same optimal length. Nodes with less than 50% support are collapsed as polytomies.