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.
Table 1.
Candidate Operon Partners Identified in the C. elegans Ribosomal Proteome Gene Set and Tested in B. malayi
Table 2.
Spacing in base pairs between Genes in Conserved Operonic Structures in Nematodes
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.
Table 3.
SL Usage in Downstream Genes Found in Operons in Different Nematode Species
Table 4.
Nematode SL Sequences
Figure 4.
(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.