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MBE Advance Access originally published online on June 22, 2005
Molecular Biology and Evolution 2005 22(10):2048-2054; doi:10.1093/molbev/msi200
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© The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org

Research Article

Spliced-Leader trans-Splicing in Freshwater Planarians

Ricardo M. Zayas1, Tyler D. Bold1 and Phillip A. Newmark

Department of Cell and Developmental Biology, University of Illinois at Urbana, Champaign

E-mail: pnewmark{at}life.uiuc.edu.


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two ~35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of ~110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing.

Key Words: spliced-leader RNA • trans-splicing • planarian • Platyhelminthes • Schmidtea mediterranea • Dugesia japonica


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Spliced-leader trans-splicing is an unconventional form of mRNA processing in which an independently transcribed leader sequence is appended to the 5' end of pre-mRNAs (Davis 1996Go; Nilsen 2001Go). It was first observed in the kinetoplastid protozoan, Trypanosoma brucei (Campbell, Thornton, and Boothroyd 1984Go; Kooter, De Lange, and Borst 1984Go; Milhausen et al. 1984Go); subsequent work identified trans-splicing in representatives from widely divergent phyla, ranging from nematodes, Platyhelminthes (flatworms), and euglena to cnidarians, chordates, and most recently, rotifers (Krause and Hirsh 1987Go; Rajkovic et al. 1990Go; Tessier et al. 1991Go; Stover and Steele 2001Go; Vandenberghe, Meedel, and Hastings 2001Go; Ganot et al. 2004Go; Pouchkina-Stantcheva and Tunnacliffe 2005Go). In trypanosomes all mRNAs are substrates for the trans-splicing reaction; in the metazoans studied to date, there is a wide range in the proportion of mRNAs that are trans-spliced, from a small subset to nearly all transcripts (Davis 1996Go). trans-Splicing has been shown to enhance translational efficiency in Ascaris (Maroney et al. 1995Go), and the spliced-leader (SL) sequence (and/or its cap structure) mediates polysome association in Leishmania (Zeiner, Sturm, and Campbell 2003Go). trans-Splicing is also involved in the processing of operon-derived polycistronic transcripts into individual mRNAs (Blumenthal 1995Go; Lee and Sommer 2003Go; Ganot et al. 2004Go). However, the precise function of trans-splicing in RNA metabolism remains unclear; it is not understood why some transcripts are trans-spliced and others are not nor is there a clear functional correlation between the mRNA targets of SL addition and the proteins that they encode. Furthermore, the evolutionary history of trans-splicing remains an open question: is it a derived or ancestral feature (Nilsen 2001Go; Stover and Steele 2001Go; Vandenberghe, Meedel, and Hastings 2001Go)? Clarification of the above questions will require the identification of additional organisms that utilize this unusual mode of RNA processing.

While analyzing a collection of expressed sequence tags (ESTs) from a free-living flatworm, the freshwater planarian Schmidtea mediterranea (R. M. Zayas, A. Hernández, B. Habermann, Y. Wang, and P. A. Newmark, in preparation) we noticed a stretch of ~35 nt that was shared between the 5' ends of hundreds of different EST clones. Here we present evidence that these sequences are derived from trans-splicing of SL RNAs to a population of transcripts in S. mediterranea. We also show that similar SL sequences are found in another planarian, Dugesia japonica, providing further evidence that trans-splicing may be an ancestral feature within the Platyhelminthes. The planarian's experimental accessibility, susceptibility to RNAi, ease of culture, and the forthcoming genome sequence of S. mediterranea provide another model organism in which to study the function(s) of trans-splicing.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Informatics
Potential trans-spliced transcripts were identified by BlastN (Stand-alone BLAST 2.2.8 [Altschul et al. 1997Go]) searches of a database containing assembled ESTs from the hermaphroditic strain of S. mediterranea (R. M. Zayas, A. Hernández, B. Habermann, Y. Wang, and P. A. Newmark, in preparation) using the planarian SL sequence. In addition, we searched a local database of whole-genome shotgun sequence reads from the S. mediterranea genome sequencing project (Washington University Genome Sequencing Center) downloaded from the National Center for Biotechnology Information (NCBI) Trace Archives (http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?). Paludicola sequences deposited in Genbank were retrieved and queried for the SL sequence by BlastN. The top hits were examined manually to verify that they corresponded to the 5' end of the sequence.

trans-Spliced cDNAs were examined by BlastX against the nonredundant protein database at NCBI. Those with significant matches were classified further by Gene Ontology (GO), using terms in the Biological Process category based on the closest annotated homolog (Ashburner et al. 2000Go; Gene Ontology Consortium 2001Go). RPS-Blast (Marchler-Bauer et al. 2005Go) was used to query the conserved domain database for conserved domains encoded by the predicted products of trans-spliced RNAs. All ESTs containing the Smed SL sequences have been deposited in GenBank; accession numbers for these sequences are available as a supplementary file (Supplementary Material online).

RNA Isolation
Total RNA was isolated from a clonal line (CIWsx2) of the hermaphroditic strain of S. mediterranea. Worms were placed on ice in RNAlater (Ambion, Austin, Tex.), and then Totally RNA (Ambion) was used to extract RNA as recommended by the supplier. Total RNA obtained in this manner was LiCl precipitated prior to use.

Rapid Amplification of cDNA Ends
Rapid amplification of cDNA ends (RACE) experiments were performed using FirstChoice RLM-RACE (Ambion). For 5' RACE gene-specific primers were designed from S. mediterranea genomic DNA using the predicted SL RNA sequence. The reverse primers used were 5'-CAAGTGACTGTCAAAAATTAACC-3' and 5'-CTAATGTTGGATAACGGTCC-3' for the outer and inner amplification reactions, respectively. For SL 3' RACE, poly(A) tails were added to 10 µg S. mediterranea total RNA using yeast poly(A) polymerase (Ambion); first-strand cDNA was then synthesized from the polyadenylated RNA. The gene-specific forward primer used for 3' RACE was 5'-GACGGTCTTATCGAAATC-3'.

An ~70-bp fragment obtained from the second round of 5' RACE amplification and an ~100-bp fragment obtained from 3' RACE were gel purified using Qiaex II resin (Qiagen, Valencia, Calif.), cloned into pCR II T-A cloning vector, and transformed into Oneshot TOP10F' competent cells (Invitrogen, Carlsbad, Calif.). Clones were checked for inserts by colony polymerase chain reaction; plasmid DNA was then purified by miniprep (Wizard Plus SV minipreps, Promega, Madison, Wisc.). Inserts were sequenced using the standard Big Dye 3.1 sequencing reaction (ABI, Foster City, Calif.) with the addition of 0.7 M betaine (Sigma, St. Louis, Mo.) and were analyzed using Sequencher 4.2.2 (Gene Codes Co., Ann Arbor, Mich.).

Northern Blot Analysis
Total RNA (10 µg) was separated in a formaldehyde/agarose gel. After electrophoresis, RNA was capillary transferred to Hybond N+ nylon membrane (Amersham, Piscataway, N.J.) and UV cross-linked to the membrane using a Spectrolinker XL-1500 (Spectronics Corporation, Westbury, N.Y.). The sequence of the oligonucleotide probe was 5'-GACGGTCTTATCGAAATCTATATAAATC-3'; 100 {rho}mol were 3'-end labeled with digoxigenin-ddUTP (Roche, Indianapolis, Ind.) using terminal transferase (Roche). Hybridizations were carried out in DIG Easy Hyb (Roche) for 18 h at room temperature. The blot was washed twice in 2 x saline sodium citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) and twice in 0.5 x SSC/0.1% SDS at room temperature. CDP-Star (Roche), a chemiluminescent substrate for alkaline phosphatase, was used to detect hybridized probe. The blot was incubated in 1% Blocking Reagent (Roche) in maleic acid buffer (MAB; 0.1 M maleic acid, 0.15 NaCl, pH 7.5) containing 1:20,000 Anti-Digoxigenin-AP (Roche), washed in MAB/0.3% Tween 20, equilibrated in detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 7.5), and then incubated in CDP-Star working solution for 5 min before exposure to X-ray film.

RNA Secondary Structure Modeling and Sequence Alignments
The SL sequences derived from RACE experiments were modeled using web-based mfold 2.3 (Zuker 2003Go). Folding was performed at 18°C and 22°C (the temperatures at which the planarians are reared in the laboratory) as well as 37°C with the constraint that the putative Sm-binding site (AAUUUUUGA) remained single stranded. To generate graphical output, the mfold predictions were imported into RnaViz 2.0 (De Rijk, Wuyts, and De Wachter 2003Go). ClustalW 1.8 (Thompson, Higgins, and Gibson 1994Go; Chenna et al. 2003Go) was used to produce multiple sequence alignments, and graphical display of the output was performed using Boxshade 3.21.


   Results and Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Supplementary Material
 Acknowledgements
 References
 
Identification of Putative SL Sequences in S. mediterranea ESTs
To study the process of epigenetic germ cell specification, we have generated an EST collection from the hermaphroditic strain of the planarian S. mediterranea. This collection of ~27,000 ESTs assembles into ~10,000 putative transcripts, ~6,500 of which are present in contiguous sequences (contigs) of two or more ESTs (R. M. Zayas, A. Hernández, B. Habermann, Y. Wang, and P. A. Newmark, in preparation). While comparing these sequences to an EST collection generated from the asexual strain of the same species (Sánchez Alvarado et al. 2002Go), we noticed that hundreds of transcripts from the hermaphroditic strain shared the 5'-most ~35 nt with the 5' ends of ~25 ESTs from the asexual planarian strain. The simplest explanation for this shared 5' sequence was that it resulted from a trans-splicing event in which an SL sequence was appended to numerous different transcripts.

To identify the potential targets of SL addition within our EST collection we used the shared 35-nt sequence to query the assembled ESTs using BlastN. This search identified 320 putative targets of trans-splicing or ~3.2% of the transcripts represented in the EST collection; this number likely underestimates the frequency of the occurrence of the putative SL sequence because its identification requires full-length or nearly full-length cDNAs. Closer inspection of the putative SL sequences revealed two distinct types: one present on 304 of the assembled sequences and another present on 16 of the sequences. Because of its abundance the former will be referred to as Smed SL-1 (fig. 1A), whereas the less prevalent form will be called Smed SL-2 (fig. 1B); they differ from each other by 2 nt (fig. 1A and B). Upon inspection of the contigs that contained SL-2, we found four in which either SL-1 or SL-2 was observed at the 5' end of the aligned ESTs; an additional eight SL-2–containing ESTs were found aligned in contigs with either SL-1 or putative 5' cis-sequences (see examples in fig. 1C in which SL-1, SL-2, and cis-sequences are all represented by ESTs). Examination of genomic DNA sequences encoding representatives of the potentially trans-spliced mRNAs revealed that the putative SL sequences were not present in the vicinity of the transcribed sequences; rather, potential splice acceptor sites were identified at the presumptive site of SL addition (fig. 1C). These results are all consistent with trans-splicing appending SL sequences to a sizable population of planarian transcripts; furthermore, it appears that mRNAs encoded by the same gene may receive either SL sequence. It will be of interest to determine if the utilization of different SL sequences has any functional significance, for example, whether they play roles in distinct cell types or at different stages of development.



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  		FIG. 1.— SL sequences from Schmidtea mediterranea. Alignments of representative EST clones containing Smed SL-1 (A) or SL-2 (B); differences between the SL sequences are shown in bold lowercase letters. The accession numbers and closest database match of the predicted products are indicated to the right. (C) Alignments of ESTs and corresponding genomic sequences reveal transcripts containing either 5'-cis sequences, SL-1, or SL-2, all of which are depicted below the genomic sequence. In all panels the SL sequences are underlined and putative start sites are shaded gray (sequences lacking a highlighted ATG have predicted start sites 3' to the indicated sequences). In (C) the spliced intron is indicated in lowercase and the splice acceptor dinucleotide (ag) is marked in bold; the genomic sequences are labeled with their Trace Archive ID (TI) numbers, and the EST accession numbers are indicated.

 
Characterization of Planarian SL RNAs
RNA blot analysis of total RNA hybridized with an antisense oligonucleotide to the predicted SL sequence revealed a prominent band of ~100 bases as well as a smear ranging in size 0.3–8 kb (fig. 2A). This result is consistent with a heterogeneous population of RNAs being the recipient of SL trans-splicing, with the SL derived from a small RNA. 5' and 3' RACE were performed to characterize the termini of the ~100-base SL RNA (see Materials and Methods). These sequences were then compared with genomic SL sequences identified by BlastN analysis of S. mediterranea whole-genome shotgun reads available via the NCBI Trace Archives. Two distinct sequences were identified that corresponded to SL-1 and SL-2 and that matched the sequences obtained by RACE (fig. 2B): SL-1 RNA is 107 bases in length and SL-2 is 106 bases long; both produce a 42-base spliced leader. Additional genomic sequences encoding potential SL-1-type RNAs were identified from the whole-genome shotgun reads. In total, we identified seven distinct genomic contigs that contain SL sequences, one of which contains three distinct SL repeats; an alignment of these sequences is shown in figure 2C. None of these sequences appears to be located within 5S RNA gene repeats (not shown), consistent with observations of SL genes in other flatworms (Davis 1997Go; Brehm, Jensen, and Frosch 2000Go).



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  		FIG. 2.— Characterization of Smed SL RNAs. (A) RNA blot of Schmidtea mediterranea total RNA hybridized with a digoxigenin-labeled probe corresponding to the trans-spliced region of SL. The size markers are indicated to the left. (B) Alignment between SL genomic sequences (top) and the longest obtained RACE products for each SL RNA. The trans-spliced sequences are underlined, and the introns are depicted in lowercase. A total of 8/10 5' RACE clones begin at the first G residue shown; arrowheads mark the starting points of two additional 5' RACE clones. The accession numbers for Smed SL-1 and SL-2 are AY949619 and AY949620, respectively. (C) Sequence alignments between all of the identified SL-encoding genomic sequences; SL-1– and -2–encoding sequences are shown on the last two lines. The Trace Archive ID (TI) numbers containing these sequences are shown to the left. *Contig20 is contained within TI 313975152 and 374117748; contig11b is contained within TI 542833743 and 545990798. Identical residues are shaded black; the extent of the SL sequence is marked by the arrowheads.

 
Secondary structure predictions for the Smed SL sequences were generated using mfold version 2.3 (Zuker 2003Go) as described in Materials and Methods (fig. 3A). Mfold predicted single structures for both Smed SL-1 and -2; raising the temperature gave identical results with slightly less favorable free energy values. There were minor differences in the predicted secondary structures of SL-1 and -2 (fig. 3A), but the conserved features observed in most other SL RNAs (Bruzik et al. 1988Go) were present in both. Three stem-loop structures are predicted, the first of which contains the SL sequence and the two additional stem loops flank the Sm-binding sequence (fig. 3A). Sequence alignments between the Smed SL RNAs and those derived from other flatworms reveal conserved sequences toward the 5' end of the SL, at the SL/splice donor junction (fig. 3B, arrowhead), and at the putative Sm-binding site (fig. 3B, underlined) (Davis 1997Go). In all of these flatworm SL RNAs, the splice donor sequence is preceded by an AUG; this start codon provides a potential role for the flatworm SL sequences in regulating translational initiation (Davis 1997Go; Brehm, Jensen, and Frosch 2000Go; Brehm et al. 2002Go). Further work will be required to demonstrate that the SL-encoded AUG can be used as a bona fide translational start site.



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  		FIG. 3.— Secondary structures of Smed SL RNAs and conservation of flatworm SL sequences. (A) Predicted secondary structures of the planarian SL RNAs generated by mfold (Zuker 2003Go) and diagrammed using RnaViz (De Rijk, Wuyts, and De Wachter 2003Go). The stem-loops are numbered 1–3. The splice sites are indicated by arrowheads, and the Sm-binding region is boxed. (B) Alignment between SL RNAs from Schmidtea mediterranea and other flatworms. The conserved splice junction is indicated by an arrowhead, and the Sm-binding domain is underlined. (C) SL sequences from the planarian Dugesia japonica aligned with the Smed SL RNAs; the single nucleotide difference from Smed SL-2 is indicated in bold lowercase letters. The Dugesia SL sequences are underlined, and putative start codons are shaded gray; accession numbers are indicated to the right.

 
Conservation of SL Sequences in D. japonica
To address the question of whether trans-splicing was more widespread within the freshwater planarians, we used the SL sequence to query Paludicola (freshwater planarian) sequences in GenBank. In addition to identifying 30 SL-containing ESTs from the asexual strain of S. mediterranea, we found 72 ESTs from the planarian D. japonica (Mineta et al. 2003Go) that harbored SL sequences (fig. 3C). There appear to be at least two distinct SL forms in D. japonica: one identical to Smed SL-1 and one with a single base mismatch to SL-2 (fig. 3C). The identification of SL sequences within two different species of freshwater planarians further strengthens the hypothesis that trans-splicing is an ancestral trait of the Platyhelminthes (Davis 1997Go); however, a limited number of flatworm groups has been studied to date (Davis 1997Go; Brehm, Jensen, and Frosch 2000Go; Brehm et al. 2002Go; this work), the most basal of which are the polyclads (fig. 4). Thus, it will be important to examine whether the basal members of the flatworm lineage (e.g., Catenulida and Macrostomida) also carry out trans-splicing. Small-scale collections of ESTs from these organisms should help address this question.



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  		FIG. 4.— Phylogenetic distribution of trans-splicing in the Platyhelminthes. The lineages where trans-splicing has been found are indicated in bold uppercase text in this consensus phylogeny of the Platyhelminthes generated by Baguñà and Riutort (2004)Go. Dashed lines indicate uncertain relationships. A clade comprising the parasitic genera Icthyophagha, Notentera, Urastoma, and Kronborgia is abbreviated INUK; not all members of the Neodermata are depicted here.

 
Annotation of trans-Spliced mRNAs
To examine potential functional relationships between the products encoded by trans-spliced mRNAs in planarians, we used these sequences as queries in BlastX searches against the nonredundant protein database (nr, NCBI). Of 320 trans-spliced sequences from the hermaphrodite S. mediterranea, 242 had significant (E < 1 x 10–4) hits to the nr database. Based on the closest annotated homolog, ESTs were assigned GO terms in the Biological Process category; 110 ESTs could be assigned to a Biological Process (Supplementary Spreadsheet 1, Supplementary Material online), and the major categories are summarized in table 1. We found that there were no obvious trends in the predicted biological functions of the products of trans-spliced mRNAs, similar to what has been described in other flatworms (Davis et al. 1995Go; Brehm, Jensen, and Frosch 2000Go; Brehm et al. 2002Go). We further examined the products of trans-spliced mRNAs by searching them for conserved protein domains (see Materials and Methods). A total of 185 ESTs were assigned domains with ~149 different domains represented (Supplementary Spreadsheet 1, Supplementary Material online). Similar to the GO annotation, the distribution of domains suggests that trans-splicing is widespread and not specific to particular gene families or functional categories.


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  		Table 1 Gene Ontology Terms Associated with trans-Spliced mRNAs in Hermaphroditic Schmidtea mediterranea

 
In this report we have provided evidence for trans-splicing of SL RNAs in freshwater planarians. Planarians are best known for their remarkable regenerative abilities (Reddien and Sánchez Alvarado 2004Go), and there has been a revival in their use as models to study regeneration (Newmark and Sánchez Alvarado 2002Go). This revival has prompted the development of a substantial toolkit for studying these organisms, including whole-mount in situ hybridization (Umesono, Watanabe, and Agata 1997Go), RNA interference (Sánchez Alvarado and Newmark 1999Go), collections of thousands of ESTs (Sánchez Alvarado et al. 2002Go; Mineta et al. 2003Go; R. M. Zayas, A. Hernández, B. Habermann, Y. Wang, and P. A. Newmark, in preparation), large-scale RNAi screens (Reddien et al. 2005Go), and the upcoming genome sequence of S. mediterranea. These tools should be helpful in characterizing the role(s) of trans-splicing in planarians.


   Supplementary Material
TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
Acknowledgements
 References
 
The complete Gene Ontology term annotation and conserved protein domain assignment results are available as a supplementary spreadsheet (Zayas et al-S1.xls). The accession numbers for all of the trans-spliced ESTs reported here are also available as a supplementary file (SmedSL_accession.txt). The supplementary spreadsheet 1 and the supplementary file are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).


   Acknowledgements
TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
References
 
We would like to thank Francesc Cebrià and Richard Davis for helpful comments on the manuscript, Bianca Habermann for annotation of planarian EST sequences, the Washington University Genome Sequencing Center and the National Human Genome Research Institute for shotgun sequencing the S. mediterranea genome, as well as Jeffrey Haas and Phil Anders for help with computer analysis. R.M.Z. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This work was supported by an NSF CAREER Award (IBN-0237825) and National Institutes of Health R01 HD-43403 to P.A.N. P.A.N. is a Damon Runyon Scholar supported by the Damon Runyon Cancer Research Foundation (DRS 33-03).


   Footnotes
 
1 These authors contributed equally to this work. Back

Billie Swalla, Associate Editor


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Accepted for publication June 10, 2005.


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