Operons are a conserved feature of nematode genomes

Abstract

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and "spliced leader" (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

Keywords: SL trans-splicing; nematode genome; operons.

Figure 1

Evidence for the existence of an enoplean operon. (A) Schematic showing the genomic organization of zgpa-1, dif-1, and aph-1 in selected nematodes mapped onto their phylogenetic relationships. Arrows represent genes, and the gray lines represent the intercistronic regions (ICRs). Numbers above the ICRs represent the distances, in base pairs, between the stop and start codons of the upstream and downstream genes, respectively. The C. elegans operon numbers are given where appropriate. Fractions below the T. spiralis and T. muris genes represent the proportion of cDNAs derived from those genes that begins with a spliced leader sequence (see also Table S2). In C. elegans, the three genes are part of different operons. * indicates the distance between genes on chromosome IV. (B) Detecting polycistronic RNAs derived from the zgpa-1∼dif-1∼aph-1 operon in enoplean nematodes. The exon–intron structures of the amplicons used to identify polycistronic RNAs are shown, with exons represented by boxes (shaded to identify the genes from which they are derived using the same color coding that was used in A. The intercistronic regions are represented by cream-colored boxes. The positions of the SL trans-spliced 3′ splice sites are indicated. The length of each cDNA is indicated.

Figure 2

T. muris SL sequences and SL RNA structure. (A) Tmu-SL1–13 genes were identified using a combination of cDNA sequencing and bioinformatics tools as described in Materials and Methods. Tmu-SL12 was found by 5′ RACE trans-spliced to nuaf-3 mRNA, and Tmu-SL13 was found trans-spliced to aph-1 mRNA. In the alignment, only the SL sequences are shown. T. muris SL sequences were manually aligned and conserved groups are countershaded. C. elegans SL1 and SL2 and the previously identified P. punctatus SL sequences were included for comparison. (B) The intron of Tmu-SL2 was experimentally identified and also found in the genome sequence. The proposed secondary structure was produced using M-fold (Zuker 2003). The SL sequence is shown in outline font and the putative Sm sequence motif is countershaded.

Figure 3

Evidence for an evolutionarily conserved nematode operon. (A) The structure of the cpt-2-nuaf-3 genomic regions from a range of nematode species mapped onto the nematode phylogeny. Genes are represented by arrows, and the gray lines represent the intercistronic regions (ICRs). Numbers above the ICRs represent the distances, in base pairs, between the stop and start codons of the upstream and downstream genes, respectively. The C. elegans operon numbers are given where appropriate. Fractions below the T. spiralis and T. muris genes represent the proportion of cDNAs derived from those genes that begins with a spliced leader sequence. (B) Detecting polycistronic RNAs derived from the cpt-2∼nuaf-3 operon in T. spiralis. The exon–intron structures of the amplicons used to identify polycistronic RNAs are shown, with exons represented by boxes (shaded to identify the genes from which they are derived using the same color coding that was used in A. The region removed during operon processing is represented by cream-colored boxes. The positions of the SL trans-splice 3′ spliced sites are indicated.

Figure 4

Processing of a synthetic operon containing the T. spiralis cpt-2∼nuaf-3 intercistronic region in C. elegans. (A) Schematic of the structure of sur-5::gfp∼mCherry synthetic operon construct containing the Tsp-cpt-2∼nuaf-3 intercistronic region. The sequence immediately downstream from the Tsp-cpt-2 3′-UTR is shown, illustrating the presence of the Ur motif and the trans-splice acceptor site. Strain PE613 contains an identical construct, but with the ICR replaced with that from between Cel-cpt-2 and prx-14. (B) Detection of operon transcript processing by SL2 trans-splicing. mCherry transcripts trans-spliced to SL2 were detected by reverse transcription of RNA prepared from either PE612 or PE613 animals followed by PCR with an SL2 primer and a primer located in the mCherry coding region (+RT). Primers amplifying gpd-1 were included to control for sample variation. gpd-1 genomic DNA was detected in the −RT control reaction (*) and SL2-ZK1236.7a is a minor product detected in the +RT reactions. Reactions with RNA isolated from N2 wild-type animals were included as control. M is a DNA size standard. (C) Alignment of transgene sequences and SL2-mCherry transcripts confirming correct splice site usage. The beginning of the mCherry open reading frame is countershaded black, the NheI cloning site is countershaded gray, and the SL2 sequences are underlined.

Figure 5

Evolutionary relationship between snRNPs associated with SL trans-splicing. Unrooted PhyML tree showing the relationship between sna-1 and sut-1 homologs identified in selected nematodes. Genes were named on the basis of their C. elegans homologs. The following species-specific prefixes were used: Aca, Angiostrongylus cantonensis; Asu, Ascaris suum; Bma, Brugia malayi; Cel, C. elegans; Cbr, C. briggsae; Cre, C. remanei; Hco, Haemochus contortus; Llo, Loa loa; Nam, Necator americanus; Rcu, Romanomermis culicivorax; and Wba, Wucheria bancrofti. The numbers at each node are approximate likelihood ratio test statistics.