SL2-like spliced leader RNAs in the basal nematode Prionchulus punctatus: New insight into the evolution of nematode SL2 RNAs

Abstract

Spliced-leader (SL) trans-splicing has been found in all molecularly characterized nematode species to date, and it is likely to be a nematode synapomorphy. Most information regarding SL trans-splicing has come from the study of nematodes from a single monophyletic group, the Rhabditida, all of which employ SL RNAs that are identical to, or variants of, the SL1 RNA first characterized in Caenorhabditis elegans. In contrast, the more distantly related Trichinella spiralis, belonging to the subclass Dorylaimia, utilizes a distinct set of SL RNAs that display considerable sequence diversity. To investigate whether this is true of other members of the Dorylaimia, we have characterized SL RNAs from Prionchulus punctatus. Surprisingly, this revealed the presence of a set of SLs that show clear sequence similarity to the SL2 family of spliced leaders, which have previously only been found within the rhabditine group (which includes C. elegans). Expression of one of the P. punctatus SL RNAs in C. elegans reveals that it can compete specifically with the endogenous C. elegans SL2 spliced leaders, being spliced to the pre-mRNAs derived from downstream genes in operons, but does not compete with the SL1 spliced leaders. This discovery raises the possibility that SL2-like spliced leaders were present in the last common ancestor of the nematode phylum.

FIGURE 1.

P. punctatus SL sequences are related to SL1 and SL2. (A) A simplified nematode phylogeny based on ribosomal RNA sequences showing clades I-V (Holterman et al. 2006; Meldal et al. 2007). Species indicated are Caenorhabditis elegans, Aphelenchus avenae, Ascaris suum, Prionchulus punctatus, and Trichinella spiralis. (B) Alignment of P. punctatus Ppu-SL1 to Ppu-SL6 spliced leader sequences with C. elegans SL1 and SL2 spliced leaders. Conserved sequence elements outside the splice junction are boxed in gray. (C) Proposed secondary structures of Ppu-SL2 and Ppu-SL3.1 RNAs (GenBank entries GU936306 and GU936307) produced using M-fold (Zuker 2003). The SL sequence is shown outlined, and the putative Sm sequence motif is underlined.

FIGURE 2.

Ppu-SL2 RNA expressed in C. elegans is trans-spliced to SL2 accepting transcripts. (A) Schematic of the expression construct. Ppu-SL2 is expressed under the control of the C. elegans U2.3 snRNA promoter, and sls-1 terminator in strains PE535/536. (B) Detection of trans-splicing of Ppu-SL2 RNA. Total RNA prepared from strains PE535/536 was reverse transcribed. cDNA synthesis was tested by amplification of act-1; trans-splicing of Ppu-SL2 to Y67H2A.5 transcripts was detected using a gene-specific primer in combination with the Ppu-SL2 primer. In a control reaction (−RT), the reverse transcriptase was omitted from the cDNA synthesis reaction. PCR reactions were analyzed by agarose gel electrophoresis (shown in inversed colors), and PCR products were analyzed by sequencing. The 5′ end of the sequence including the Ppu-SL2 primer sequence is shown. Critical residues that allow the distinction between trans-splicing to Ppu-SL2 and to SL1 or SL2 are indicated with arrows, and the Y67H2A.5 transcript region is boxed black, with the beginning of the open reading frame in italics. The SL2 splice acceptor site is indicated (Δ). (C) Trans-splicing of Ppu-SL2 to tct-1, rla-1, and rps-3 transcripts in PE536. Trans-splicing to SL1 or SL2 or Ppu-SL2 RNAs was detected using gene-specific primers in combination with SL1, SL2, or Ppu-SL2 primers. Control PCR amplifications were performed with mock cDNA preparations performed without reverse transcriptase (−RT). Amplification products were identified by DNA sequencing. M indicates marker lane, in base pairs (bp). The acceptor sites for SL2 and Ppu-SL2 are indicated in the genomic tct-1 and rla-1 sequences (▲).

FIGURE 3.

Ppu-SL2 is specifically trans-spliced to the SL2 acceptor site of pkc-1. (A) Schematic showing the first (kup-1) and second (pkc-1) gene in operon CEOP5312 with SL1 and SL2 splice acceptor sites. Note that the SL2 acceptor site and the two first exons of pkc-1 are not currently annotated in WormBase. (B) Trans-splicing of Ppu-SL2, SL1, and SL2 RNAs to kup-1 and pkc-1 in PE536 was detected using the combination of gene-specific primers with SL1, SL2, or Ppu-SL2 primers indicated. Control PCR amplifications were performed with mock cDNA preparations performed without reverse transcriptase (−RT). Amplification products were identified by DNA sequencing. The SL1 and SL2 acceptor sites (Δ) are indicated in the sequence of the Ppu-SL2/pkc-1 fragment shown. The first two exons are boxed in gray; the following exons, in black. The beginning of the open reading frame is italicized. M indicates marker lane, in base pairs (bp).