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Originally published In Press as doi:10.1074/jbc.M006091200 on September 5, 2000

J. Biol. Chem., Vol. 275, Issue 49, 38311-38318, December 8, 2000
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mRNA Trans-splicing in the Human Parasitic Cestode Echinococcus multilocularis*

Klaus BrehmDagger, Katharina Jensen, and Matthias Frosch

From the Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany

Received for publication, July 11, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An identical 36-nucleotide exon was identified at the 5' termini of different mRNAs from the cestode Echinococcus multilocularis. We provide evidence that this exon constitutes a new spliced leader (SL) that is obviously trans-spliced to echinococcal pre-mRNAs, donated by a non-polyadenylated, trimethylguanosine-capped SL-RNA of 104 nucleotides. Sequence comparisons indicated that cestode and trematode SLs are likely to be derived from a common ancestor gene. No conservation was, however, observed concerning the spectrum of mRNAs that is trans-spliced in cestodes and trematodes, indicating that trans-splicing of a particular flatworm mRNA is not correlated with the function of the encoded protein. We also show that the echinococcal gene elp, encoding a member of the ezrin/radixin/moesin protein family, is expressed via two alternative transcripts, spliced either cis or trans at an identical splice acceptor site. This was accompanied by the formation of different elp primary transcripts, harboring a complete or a truncated upstream intron, which supports the hypothesis that alternative cis/trans-splicing depends on the presence or absence of an upstream splice donor site. A putative SL gene was also identified on chromosomal DNA of Echinococcus granulosus, indicating widespread utilization of trans-splicing in the genus.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Trans-splicing is a mechanism of mRNA processing that involves the fusion of exons from independent primary transcripts to form a mature mRNA (for recent reviews, see Refs. 1-5). In the most common form of trans-splicing, called SL1 trans-splicing, a small "mini-exon" (or SL) is added to the 5' ends of pre-mRNA molecules, eventually forming the 5'-terminal exon of the mature mRNA. In all cases investigated so far, the SL donor molecule is a small, non-polyadenylated nuclear RNA (the SL-RNA) with structural properties similar to the snRNAs that function in "conventional" cis-splicing (1-5). SL trans-splicing was first described in kinetoplastid protozoans where all mature mRNAs contain an identical leader sequence at the 5' end (6). Among metazoans, SL trans-splicing is known for several parasitic and non-parasitic nematodes (1-6) and, in the phylum Platyhelminthes, for parasitic trematodes (7-9) and free-living turbellarians (9). In contrast to kinetoplastids, where trans-splicing is the predominant form of splicing, transcripts of metazoans are generally spliced both cis, usually at introns within the coding region, and trans at the 5' end (1-5). A further difference is that in metazoans not all mRNAs are trans-spliced. In Caenorhabditis elegans, for example, around 60% of all transcripts contain one leader, called SL1, and about 10-15% contain an alternative mini-exon, called SL2 (1-5). Besides SL1 and SL2, three additional SLs were identified in C. elegans that are, however, less frequently found at mRNA 5' ends (10).

Although significant data has been obtained on the biochemistry of the trans-splicing mechanism itself (1-6), the biological in vivo functions of SLs are less well understood. At least one cellular mechanism that employs trans-splicing is the processing of polycistronic messages into individual coding units as demonstrated for trypanosomes and C. elegans (1-5). A role of the SL in resolving polycistronic messages has also been suggested for the trematode Schistosoma mansoni (11). However, only a fraction of around 25% of all C. elegans genes are expressed as operons, and among the monocistronically transcribed mRNAs, some are trans-spliced and some are not (3, 4). This strongly implies additional functions of SLs, and it has been proposed that they may participate in the regulation of translation, in controlling mRNA stability, or in directed transport of transcripts within the cell (1, 4, 12). So far no specific sequence motifs critical for SL function have been identified, since SLs of different phylogenetic groups are highly divergent at the level of primary sequence. A particular role for SL function in helminths could, however, have the TMG cap of trans-spliced transcripts, which derives from the donor RNA and which distinguishes these mRNAs from non-trans-spliced transcripts containing a 7-methylguanosine cap (13-15).

After the description of SLs in parasitic nematodes and trematodes, some as yet unsuccessful attempts have been made to also identify trans-splicing in the third group of medically important helminths, the cestodes (7, 9). In this study we have investigated gene expression mechanisms in the cestode Echinococcus multilocularis, the so-called fox tapeworm. The larval stage of E. multilocularis is the causative agent of alveolar echinococcosis, which is considered to be the most lethal helminthic infection in humans (16). As in the case of the closely related dog tapeworm Echinococcus granulosus (16), the causative agent of cystic echinococcosis, no information on gene regulatory mechanisms is available for E. multilocularis, and only a few genes have been characterized for both species. We now present evidence that the echinococcal gene elp, encoding a member of the ezrin/radixin/moesin family of proteins (17, 18), is expressed via two alternatively spliced transcripts, one of which contains an echinococcal spliced leader at its 5' end. We have identified several echinococcal mRNAs that are processed by trans-splicing and provide a detailed characterization of the SL encoding gene and the SL-RNA.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Organisms-- Origin and characteristics of the natural E. multilocularis isolates H-95 as well as E. granulosus larvae from an Italian bovine hydatid cyst have been described before (19, 20). E. multilocularis isolate K188 was obtained as infective eggs from the intestine of a fox from the region of Carinthia, Austria, and further propagated in mongolian jirds as described before (19).

Parasite Cultivation and Nucleic Acid Isolation-- E. multilocularis larvae were routinely kept in mongolian jirds (Meriones unguiculatus) as described (19). In vitro cultivation of metacestodes was performed according to Jura et al. (19), and protoscoleces were isolated as described in Brehm et al. (21). Chromosomal DNA and total RNA were isolated from in vivo cultivated parasite material according to previously described protocols (22). For RNA isolation from in vitro cultivated metacestodes and protoscoleces, the RNeasy-KIT (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions.

Blot Analyses and Cloning Procedures-- For Northern blot experiments, RNA samples were separated on 2% formaldehyde agarose gels or on 8% polyacrylamide, 7 M urea gels, transferred, and hybridized with 32P-labeled oligonucleotide probes as described previously (7). Cloning of PCR products and PCR-generated cDNAs was performed by TA-cloning employing the TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions.

5'- and 3'-RACE-- 5'-RACE was performed using oligo(dG)-tailing of single stranded-cDNA and subsequent PCR amplification essentially as described before (17). For 3'-RACE of the SL-RNA, total RNA was first poly(A)-tailed using poly(A) polymerase, reverse-transcribed, and then amplified using an internal primer for the SL (5'-TGC AGT TTT GTA TGG TGA GT-3') and a primer for the poly(A) tail according to a previously described protocol (23). The SL RNA 3' end was subsequently determined by sequencing of obtained PCR products.

Preparation of poly(A)+ RNA and TMG Immunoprecipitation-- For the isolation of poly(A)+ and poly(A)- RNA fractions, the Oligotex mRNA purification kit (Qiagen) was used according to the manufacturer's instructions. After repeated application of total RNA to oligo(dT) columns, both the poly(A)+ and the poly(A)- fractions were collected, precipitated, and subjected to gel electrophoresis as described above. Anti-TMG immunoprecipitation was performed as described before (24) using the monoclonal anti-TMG antibody K121 (Calbiochem).

PCR and RT-PCR Amplification-- For standard PCR amplifications, the Eurogentec (Seraign, Belgium) GoldStar Taq system was used. For the amplification of long products, the Advantage 2 PCR kit (CLONTECH, Palo Alto, CA) was employed according to the manufacturer's instructions. In RT-PCR reactions, mRNA was reverse-transcribed using Omniscript reverse transcriptase (Qiagen) and, subsequently, either PCR amplified as described above or using the One-step PCR kit (Qiagen). Detection of pre-mRNA was performed using the sensitive Titan One-step RT-PCR system of Roche Molecular Biochemicals. PCR primers were purchased from ARK Scientific (Darmstadt, Germany). Sequences of oligonucleotides described in the text were: SL-5 (5'-AAG GAC CGA TTA ACG GTG-3') and SL-3 (5'-ACC TTG CAG TTT TGT ATG-3') for cloning the SL repeat; E10-6 (5'-ATC GTA CTG TTC CTT GCT G-3'), E10-24 (5'-GAT GAT GCT CCG CAT AAT CCA-3'), E10-19 (5'-CGT TGG CAG TAT TCT CAC CG-3'), E10-31 (5'-TTC ACT TCT CGT GAA CAG TAG-3'), and E10-29 (5'-CAG TAG CCT CCT TGT TTG AG-3') for mapping the pre-mRNA start point in the 5' intron.

cDNA Library Construction-- 1 µg of total E. multilocularis RNA was reverse-transcribed as described above using primer A-37 (5'-GGC CGC ATG CCG ACT AGT ACT17-3') directed to the mRNA poly(A) tail. The resulting single-stranded-cDNA was PCR-amplified with primer A-20 (5'-GGC CGC ATG CCG ACT AGT AC-3') and primer SL-5PR (5'-CAC CGT TAA TCG GTC CTT AC-3') specific for the echinococcal SL using the Advantage 2 PCR kit (CLONTECH) and employing 20 PCR cycles (1 min at 94 °C, 1 min at 57 °C, 5 min at 68 °C) to avoid over-representation of small PCR products. The products were subsequently cloned as described above.

Competitive PCR-- Competitive PCR experiments were performed in adaptation to previously described methods (25, 26). For measuring amounts of conventionally and trans-spliced elp transcripts, one single competitor was constructed as follows: by overlap extension (27) of PCR-generated elp chromosomal and cDNA fragments, a 584-bp fragment was constructed that contained the primers EC5 (5'-GGG CTC CCG TTG ATT GCA GT-3'; see cDNA nt 11-30 in Ref. 17), specific for exon I, and E10-6 (5'-ATC GTA CTG TTC CTT GCT GG-3'), spanning the exon IV/exon V boundary (17) at its ends. Due to the presence of elp intron II (but absence of the 5' intron and intron I), this fragment was 74 bp longer than the conventionally spliced mRNA fragment between EC5 and E10-6. By further overlap extension, oligonucleotide ET5 (5'-TCC TTA CCT TGC AGT TTT GT-3'), specific for the SL, was fused to the EC5 binding site of the above fragment. The resulting fragment for ET5 and E10-6 was 94 bp longer than the corresponding fragment for the trans-spliced elp mRNA. Serial dilutions of the resulting fragment were then used in competitive PCR, with constant amounts of cDNA produced from RNA isolated from metacestodes and protoscoleces applying 30 cycles of 1 min at 94 °C, 1 min at 57 °C, 1 min at 72 °C and using the primer combinations EC5 × E10-6 for elp transcript A as well as ET5 × E10-6 for transcript B. As a control for total elp mRNA, the competitor was used for PCR experiments with primer E10-6 and E10-15 (5'-AAT AAG GTC AGG GTG ACT AC-3'; see cDNA nt 69-89 in Ref. 17), specific for elp exon III. Equimolarity of cDNA and competitor was determined by 2% agarose gel electrophoresis and ethidium bromide staining.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Alternatively Spliced elp Transcripts-- We previously characterized the chromosomal elp locus and investigated elp transcription by 5'-RACE (17). One transcript, herein termed transcript A, was identified that derived from a fusion of the non-translated 32-nt exon I to the translational start codon containing exon II, thus revealing the presence of a 386-nt intron in the 5'-non-translated region (a so-called 5' intron) (Fig. 1). In the present study, we have analyzed additional 5'-RACE clones and identified a second transcript, B, in which exon I was precisely replaced by a so far unknown 36-nt sequence (spliced leader in Fig. 1). This alternative exon showed no homology to exon I and was not present within the elp locus or in chromosomal regions of around 2 kb down- or upstream of the coding region. Sequence analysis also revealed that transcripts A and B were identical from exon II to the poly(A) addition site and that, indeed, the only difference was the alternative exon at the 5' end. Southern hybridization (28) and PCR analysis of E. multilocularis chromosomal DNA furthermore indicated that elp is present as a single copy gene per haploid genome and that the isolates investigated in this study were homozygous for the locus (data not shown). Hence, transcripts A and B are likely to be derived from the same allele.



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  		Fig. 1.   Identification of alternative elp transcripts. The chromosomal situation around the 5' intron of the E. multilocularis elp locus (from Ref. 17) together with the 5'-terminal sequences of conventionally (Transcript A) and trans-spliced (Transcript B) elp mRNAs as determined by 5'-RACE is shown. Exonic sequences are represented by capital letters, intronic and non-transcribed regions are represented by small letters. Size and location of elp exon I, exon II, and the 5' intron are indicated by lines and small open arrowheads (< >) above the nt sequence. Exon-intron boundaries are shown as double-slashes (//), and the 5' intron splice donor and splice acceptor regions are marked above the intron sequence. Sequence and location of the oligonucleotides 10-31 and 10-29 used for mapping the alternative transcriptional start point within the 5' intron are shown as arrows (--- >) below the nt sequence. Alternative 5' exons of transcript A (exon I) and B (spliced leader) are underlined, and the transition point to exon II is marked by a dash. The deduced amino acid sequence of the Elp protein N terminus, which has been confirmed by direct protein sequencing of immunoprecipitated Elp, is shown below the nt sequence in uppercase letters.

We were interested in the relative abundance of both transcripts and conducted competitive PCR experiments using upstream primers specific for exon I or the alternative exon and downstream primers specific for the exon IV/V boundary of elp. We consistently measured amounts of transcript A and transcript B in a relation of around 1:2 using parasite material isolated from M. unguiculatus as an intermediate host (not shown). Since this parasite material was composed of two different developmental stages of E. multilocularis, the metacestode and the protoscolex, we investigated whether stage-specific differences in elp expression could lead to the alternative transcripts. However, in protoscoleces manually picked from parasite material as well as in metacestode vesicles after in vitro cultivation, a constant relation of about 1:2 between transcript A and B was observed (Fig. 2).



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  		Fig. 2.   Competitive PCR experiments on alternative elp transcripts in E. multilocularis metacestodes and protoscoleces. Upon in vitro cultivation of E. multilocularis larvae (19), 50 protoscoleces (P) and 30 metacestode vesicles (mean diameter, 1 mm) (M) were manually picked, total RNA was isolated and reverse-transcribed using an oligo(dT) primer, and the resulting cDNA was diluted to 50 µl. Per PCR reaction, 1 µl of the diluted cDNA was mixed in a 30-µl final volume with 1 × 106 (lane1), 5 × 105 (lane 2), 2.5 × 105 (lane 3), 1.2 × 105 (lane 4), 6 × 104 (lane 5), and 3 × 104 (lane 6) molecules of the competitor that was constructed as described under "Experimental Procedures." For competitive PCR, the primer combinations 10-6 × EC5, specific for the exon I-containing transcript A (A) and 10-6 × ET5, specific for the SL-containing transcript B (B), were added, and 30 cycles were applied. 10 µl of each reaction were separated on a 2% agarose gel and stained with ethidium bromide. In each lane, the upper band corresponded to the competitor, and the lower band corresponded to the cDNA. Equimolarity between competitor and cDNA was reached at ~1.2 × 105 molecules for transcript A and ~2.5 × 105 molecules for transcript B in the protoscolex fraction. For the metacestode fraction, the respective values were ~2.5 × 105 for transcript A and ~5 × 105 for transcript B. Thus, a constant relation of about 1:2 between transcripts A and B was measurable in both developmental stages.

Identification of an E. multilocularis SL-RNA-- Based on the data outlined above we assumed that exon I was fused to exon II by conventional cis-splicing and that the alternative 36-nt exon derived from trans-splicing involving an echinococcal SL-RNA. This hypothesis was tested by Northern blot experiments using antisense oligonucleotides specific for exon I (probe A) and the 36-nt exon (probe B) as probes. As expected, probe A identified a single band of ~2.1 kb (Fig. 3A) that corresponded to the previously determined size of the elp transcript (20). Probe B, on the other hand, produced a hybridization smear for polyadenylated transcripts ranging between 500 nt and several kb (Fig. 3A). In addition, probe B produced a strong hybridization signal for a non-polyadenylated RNA of ~100 nt (Fig. 3, A and B) that was present in the TMG-precipitable RNA fraction but absent in the non-TMG fraction (Fig. 3B). These considerable similarities to trans-splicing elements of nematodes and trematodes (see above) led us to conclude that the 36-nt alternative exon of elp constitutes a SL from E. multilocularis that was donated to a set of different transcripts by a SL-RNA of around 100 nt. It should be noted that results similar to that described above were also obtained when RNA from protoscolex or metacestode was analyzed (not shown). Hence, at least for these two developmental stages, no stage-specific patterns of SL expression or trans-splicing were observed.



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  		Fig. 3.   Northern blot analysis of transcripts containing the SL. A, analysis of poly(A)+ and poly(A)- fractions. E. multilocularis was cultivated in M. unguiculatus, and parasite RNA was isolated. 10 µg of total RNA (lanes 1 and 4) and equivalent portions of poly(A)- (lane 2) and poly(A)+ RNA (lane 3) were separated on a 2% formaldehyde gel, blotted, and hybridized with 32P-labeled oligonucleotide probes specific for elp exon I (probe A; lane 4) or the SL (probe B; lanes 1, 2, and 3). Molecular size marker bands are indicated on the left. Probe A hybridized to a single 2.1-kb fragment. Probe B strongly hybridized to a non-polyadenylated RNA of ~100 nt and to a smear of polyadenylated transcripts between 500 nt and >4 kb. B, analysis of anti-TMG immunoprecipitated RNA. 5 µg of total RNA (lane 1) and equivalent portions of RNA in the pellet (lane 2) or the supernatant (lane 3) after immunoprecipitation with the anti-TMG cap-specific antibody K121 were separated on a 8% polyacrylamide, 7 M urea gel, transferred, and probed with a 32P-labeled, anti-SL specific oligonucleotide (probe B). Size markers at the left and right were produced from differently sized PCR products containing the SL sequence after in vitro transcription using T7 polymerase (29). Probe B strongly hybridized to a RNA of ~100 nt, which was present in the anti-TMG-precipitated fraction but absent in the non-TMG fraction.

To test whether the TMG cap deriving from the donor RNA was still present on trans-spliced echinococcal transcripts, we performed elp-specific RT-PCRs on mRNA of the TMG-precipitated fraction. In these experiments, we could clearly identify the trans-spliced transcript B of elp, whereas no amplification products were obtained for the conventionally spliced transcript A (not shown). Hence, conventionally and trans-spliced elp transcripts could be clearly distinguished by the absence or presence, respectively, of a 5' TMG cap.

Cloning and Characterization of the SL-RNA-encoding Gene-- In all organisms investigated so far, the SL-RNA-encoding genes are present on DNA fragments that are directly repeated more than 100-fold on the chromosome (6-8). Assuming that this is also the case for E. multilocularis, we chose a PCR approach to clone the SL-encoding gene, emsl, using two primers (SL-5, SL-3) that were specific for the SL but were oriented in opposite directions. After PCR amplification from chromosomal E. multilocularis DNA as a template, a 1513-bp fragment was obtained, cloned in Escherichia coli, and entirely sequenced. As expected, the 36-nt SL sequence was immediately followed by a consensus splice donor sequence (5'-GTGCGT-3') (Fig. 4A). We then used the sequence information of the SL downstream region to (i) verify the SL 5' end present on the 5'-RACE product by primer extension (not shown) and (ii) precisely map the SL-RNA 3' end by 3'-RACE (see "Experimental Procedures") to a position 104 bp downstream of the SL 5' end. A computer-generated, energy-minimized RNA secondary structure for the SL-RNA is shown in Fig. 4B. All conserved features of other SL-RNAs (4, 6, 8, 9), including three stem-loop structures, a putative Sm binding sequence between stem-loops 2 and 3, and the typical location of the SL within the first stem-loop, are present.



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  		Fig. 4.   Sequence comparison and structure of echinococcal SL RNAs. A, comparison of flatworm SL RNA gene sequences. Aligned are nt sequences from E. multilocularis (Em) and E. granulosus (Eg), determined in this study, and those from F. hepatica (Fh) and S. mansoni (Sm) taken from Davis and Hodgson (11). Nucleotides identical to that of the E. multilocularis SL RNA are marked by dots, and dashes represent gaps inserted to maximize the alignment. Additionally indicated are the length of the SL genes (in bp) and homologies to the E. multilocularis SL gene for the SL region alone and for the full-length SL RNA. B, possible secondary structure of the E. multilocularis SL RNA. The splice site is indicated by an arrow, the SL is indicated in bold letters, and the TMG cap is indicated by an asterisk. A putative Sm binding site with homology to previously presented flatworm Sm binding sequences (11) is underlined. The RNA was folded using an algorithm of Mathews et al. (30), with the constraint that the putative Sm binding sequence is single-stranded.

Sequence analysis of the 1513-bp fragment harboring emsl did not indicate the presence of putative protein-coding regions or of other snRNA-encoding genes. Furthermore, unlike nematode SL repeats, no sequences homologous to 5S rRNAs were identified in the E. multilocularis sequence.

Southern blot analysis revealed that the 1.5-kb fragment was indeed directly repeated on the E. multilocularis chromosome (data not shown). To measure the copy number, we chose a competitive PCR approach using as a reference the elp locus, which was known to be present as a single-copy gene (28). On chromosomal DNA of different natural and clinical E. multilocularis isolates, we constantly measured a relation of around 20:1 between emsl and elp (data not shown), revealing a SL gene copy number significantly lower than that previously estimated for trematodes (100-200; Refs. 7 and 8) and nematodes (100; Ref. 6).

Characterization of the SL Repeat and the SL Gene, egsl, from E. granulosus-- To investigate the possibility of trans-splicing in the dog tapeworm, we performed PCR experiments using several primers specific for the E. multilocularis SL repeat and chromosomal E. granulosus DNA as a template (not shown). These studies led to the characterization of a 1555-bp repeated element that shared 87% homology with the E. multilocularis repeat. Two nucleotide substitutions were observed for the SL-RNA, one of which was located in the SL sequence (Fig. 4A). A sequence comparison of the echinococcal SL genes and the previously characterized SL-RNAs of the parasitic flatworms S. mansoni and Fasciola hepatica is depicted in Fig. 4A. Apart from some conserved regions, particularly around the splice donor site, we could merely detect an overall homology of around 30% between the cestode and trematode SLs, although higher degrees of conservation (50-55%) were measurable for the SLs. No significant homology was observable between echinococcal SL-RNAs or SLs and those of nematode or protozoan origin.

Characterization of Additional Trans-spliced mRNAs-- To verify that the hybridization smear for polyadenylated RNAs shown in Fig. 3 did indeed represent a set of mRNAs that harbor the SL at the 5' end, a cDNA library specific for trans-spliced transcripts was constructed. After reverse transcription of E. multilocularis mRNA and PCR amplification of the single-stranded cDNA with primers specific for the poly(A) tail and for the 5' end of the SL (see also "Experimental Procedures"), a smear of products between 500 bp and ~4 kb was obtained (not shown). The PCR products were cloned in E. coli, and the recombinant plasmids of 40 randomly chosen colonies were sequenced. In all cases, the 18 bp of the SL-specific amplification primer were followed by the remaining 18 bp of the SL identified for elp, indicating that the cDNAs derived from trans-spliced mRNAs and that in all an identical SL was present. For three of the cDNAs (Em4231, Em1342, Em2222) we also determined the chromosomal situation and found consensus splice acceptor sequences immediately upstream of the mRNA SL addition site (not shown). ORFs could be identified in the inserts of 39 plasmids, and the deduced amino acid sequences of 17 plasmids revealed significant data base matches with already known proteins. Six were homologous to proteins with unknown functions. The remaining 11 are listed in Table I. Concerning the putative biological function of the encoded proteins, no common patterns were identified although three of the factors, corresponding to clones Em4231, Em3332, and Em4121, are potentially involved in splicing processes. The remaining factors are potentially involved in cellular processes as different as, for instance, glycolysis, pyrimidine metabolism, the electron transport chain, or translation initiation (see Table I).


                              
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Table I
Trans-spliced mRNAs of E. multilocularis
A cDNA library for trans-spliced mRNAs of isolate H95 was constructed as indicated under "Experimental Procedures." The inserts of 11 recombinant clones that yielded significant BLAST scores were entirely sequenced and are listed. Indicated are for each clone: cDNA size (in bp; excluding the poly(A) tail and the 36-nt SL sequence), length of the predicted ORF (in amino acids), homology (in percent identical amino acids) of the deduced amino acid sequence to the best matching amino acid sequence in databases, predicted identification according to homology, and accession numbers of best matching proteins together with their origin. Additionally shown are the 5'-terminal-nt sequences of the cDNAs, potential protein-coding regions (bold letters), and the first amino acids of predicted ORFs (below the nt sequence). The SL addition site is marked by a slash (/) with the SL 3' ATG indicated to the left. In clones Em1433 and Em1431, the 3' AUG of the SL is predicted to serve as translation start. In clones Em2342 and Em2313, the SL AUG is in-frame with the putative translation start, whereas in all others, the SL AUG is out of frame to the predicted ORF. SP, Swiss-Prot; PRF, Protein-Research-Foundation; GP, GenBank protein.

With its 3' end, the echinococcal SL provides trans-spliced mRNAs with an AUG codon that, according to the first-AUG rule (32), constitutes a potential ORF translation initiation start point. However, when analyzing the 5' ends of the above-mentioned trans-spliced echinococcal mRNAs, we found the SL-AUG in 7 of 12 cases out of frame to the actual start-AUG as predicted by sequence comparison (Table I). In three cases, including elp, the SL-AUG was present in the same reading frame as the predicted start-AUG. For elp this could possibly result in the production of two different proteins from the 5' cis- and trans-spliced transcripts (see Fig. 1). N-terminal sequencing of immunoprecipitated Elp protein, however, revealed a uniform N terminus identical to the sequence deduced from the 5' cis-spliced transcript A (not shown). We therefore conclude that in both elp transcripts the same AUG start codon is used and that the SL-AUG of transcript B does not serve for translation initiation. For two transcripts (Em1433, Em1431), on the other hand, sequence comparisons indicated that the SL-AUG codon most probably serves as the actual translation start. In both cases, translation from alternative downstream AUGs would result in products that lack a considerable part of structures that are highly conserved among the homologous members of the respective protein families.

Identification of Alternative elp Primary Transcripts-- In a final set of experiments we were interested whether alternative cis/trans-splicing at the splice acceptor site upstream of elp exon II did occur at the same pre-mRNA or whether two different primary transcripts were involved. By primer extension experiments, only the completely spliced 5' ends of transcripts A and B could be identified (data not shown), presumably because the concentration of pre-mRNA in the echinococcal RNA preparations was below detection level. We therefore chose RT-PCR using one downstream primer spanning the exon IV/exon V boundary of the elp-coding region (primer 10-6) and two different upstream primers that were located close to the 5' intron splice-donor (10-24) and splice acceptor sites (10-19), respectively. Interestingly, with the combination 10-6 × 10-19, a PCR product was obtained that, as determined by DNA sequencing, was completely spliced for the introns I, II, and III of elp but still retained the 3' part of the 5' intron (see Fig. 5). The combination 10-6 × 10-24, on the other hand, did not reveal a PCR product, even with elevated amounts of RNA. The likeliest explanation for these results is the presence of an additional transcriptional start point within the 5' intron which leads to the expression of a second elp primary transcript that lacks the 5' intron splice donor site. This indicates that cis-splicing can most probably precede trans-splicing in E. multilocularis, similar to the situation previously described for the nematode Onchocerca volvulus (33). Using additional primer combinations, we localized the alternative starting point to a region approximately between 256 and 244 nt upstream of the 5' intron splice acceptor site since the combination 10-6 × 10-29 still revealed a completely spliced fragment for the introns I-III, whereas the combination 10-6 × 10-31 gave no product (Fig. 5, Fig. 1).



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  		Fig. 5.   Mapping of a transcriptional start point within the elp 5' intron. A, schematic of the chromosomal situation around the elp 5' intron, indicating size and location of exons (white boxes) and introns (gray boxes). Oligonucleotide primers used for mapping the transcriptional start point are indicated by small arrows. B, PCR and RT-PCR analysis. 2 µg of total RNA of the E. multilocularis isolates H95 (lanes 1, 4, and 7) and K188 (lanes 2, 5, and 8) were RT-PCR-amplified in a 30-µl volume employing a sensitive one-step RT-PCR system (Titan, Roche Molecular Biochemicals) and using the primer combinations 10-31 × 10-6 (lanes 1 and 2), 10-29 × 10-6 (lanes 4 and 5), and 10-19 × 10-6 (lanes 7 and 8). 10 µl of each reaction were separated on a 2% agarose gel and stained with ethidium bromide. In addition, all PCR products were directly sequenced. As controls, the same primer combinations were used for PCR amplification of chromosomal DNA of isolate H95 (lane 3, 10-31 × 10-6; lane 6, 10-29 × 10-6; lane 9, 10-19 × 10-6). Products completely spliced for introns I, II, and III but still containing parts of the 5' intron up to the 10-29 primer binding site were obtained for both isolates (lanes 4, 5, 7, and 8). No products were obtained for 10-31 × 10-6, although this primer combination gave a clear positive signal on chromosomal DNA.

Since trans-splicing can be associated with the expression of polycistronic transcripts, we investigated similar aspects concerning elp. However, neither Northern blot hybridization experiments nor RT-PCR amplification using primers specific for elp and chromosomal upstream or downstream regions led to any indication of polycistronic pre-mRNAs, even when elevated amounts of RNA were applied (not shown). In addition, 800 bp upstream of the elp 5' intron, we identified a putative ORF encoding a protein with 86% amino acid sequence identity to nuclear proteins of Drosophila melanogaster and Homo sapiens. This reading frame was, however, transcribed in the opposite direction than elp (not shown). Up to 2 kb downstream of elp, we could not identify any additional reading frames. Taken together, these data indicate that elp is most probably expressed monocistronically.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several evidences provided in this study are clearly indicative of SL trans-splicing in E. multilocularis as follows. (i) An identical 36-nt exon was found at the 5' ends of different echinococcal mRNAs at a position where, in the respective chromosomal loci, a consensus splice acceptor site was located; (ii) the 36-nt exon was not part of these chromosomal loci but was encoded by a gene, emsl, located elsewhere on the chromosome on a directly repeated 1.5-kb fragment; (iii) the 36-nt exon formed part of a 104-nt, non-polyadenylated, TMG-capped RNA with structural characteristics of snRNAs, where it was immediately followed by a splice donor consensus site. These characteristics are highly reminiscent of SLs and SL-RNAs from kinetoplastid, nematode, and trematode origin (1-6). We conclude that the 36-nt exon is a SL from E. multilocularis and that the 104-nt RNA is the corresponding SL-RNA. A gene highly homologous to emsl, egsl, was identified on chromosomal DNA of E. granulosus, suggesting that mRNA trans-splicing is also used for gene expression in the dog tapeworm.

By the identification of SLs in the genus Echinococcus we have, for the first time, provided evidence for trans-splicing in tapeworms. Earlier studies on the distribution of trans-splicing in the phylum Platyhelminthes led to the identification of SLs in several trematode species (7, 8, 11) and in the polyclad turbellarian Stylochus zebra (11), representing two of the three major flatworm groups (34). The failure to detect similar SLs in cestodes raised questions on a possible polyphyletic origin of flatworms, the loss of trans-splicing in flatworm subgroups, or the presence of evolutionary divergent SL-RNAs (11). Our results now indicate that trans-splicing is indeed a widespread feature of the phylum, similar to the situation described for kinetoplastids and nematodes (1-6). Furthermore, due to the observed sequence homologies in their primary sequences, we suggest that all flatworm SLs identified so far derive from a common ancestor gene.

Similarities and differences of the echinococcal trans-splicing elements to that of trematodes, nematodes, and protozoa correlate with the phylogenetic position of cestodes. The Echinococcus SL-RNA displays certain homologies to that of trematodes but none to those of nematodes or kinetoplastids. The SL-encoding genes of all flatworms are located on chromosomal repeats, which, albeit variable in length and copy number, do not contain the 5 S rRNA gene, a characteristic of SL repeats from nematodes (1), euglena (35), and some trypanosomes (36). Furthermore, and typical for metazoans, not all echinococcal mRNAs are trans-spliced. Analysis of elp transcripts revealed that trans-spliced and non-trans-spliced mRNAs can even derive from one single gene locus, and recently, we have characterized beta -tubulin-encoding transcripts of E. multilocularis (21), which are exclusively expressed without spliced leader. Based on a preliminary analysis of 150 randomly chosen, full-length cDNAs (data not shown) we estimate that ~25% of all E. multilocularis mRNAs acquire the SL, a figure close to what has been suggested for trematodes (37).

Two structural features, the 5' TMG cap, which is conserved among all SLs, and the 3' AUG codon, which is specifically conserved in flatworm SLs, were also present in the echinococcal SLs. For C. elegans it has been shown that the TMG cap is still retained on the mature mRNAs after trans-splicing (14, 15), and our data on the alternatively spliced elp transcripts suggest that this is also the case for flatworms. Using an Ascaris in vitro system, evidence for a TMG cap involvement in translation initiation has been obtained (12), and recently, several isoforms of the translation initiation factor 4E (eIF4E) were identified in C. elegans that can differentiate between TMG- and 7-methylguanosine-containing transcripts (38). It will be worthwhile to investigate whether alternative translation initiation factor 4E expression also occurs in flatworms and whether this feature could be involved in translational control mechanisms of trans-spliced mRNAs. Concerning the 3' AUG codon and its possible role as a translational start point, the situation in E. multilocularis closely parallels that in F. hepatica (37). In 2 of 12 analyzed transcripts, this AUG codon obviously provided the translation start point, whereas at least in 8 a similar role could be excluded. As in the case of the TMG cap, further experiments are required to investigate whether the occasional provision of a translation start codon is the only function of the 3' AUG.

A remarkable difference between cestodes and trematodes was the variability in genes that are expressed via trans-splicing. Although the echinococcal ubiquinol-cytochrome c reductase-binding protein and fructose-bisphosphate aldolase proteins identified in this study have clear homologs in F. hepatica and S. mansoni (11, 31), the respective mRNAs are trans-spliced in E. multilocularis but not in trematodes. Further examples are the enolase encoding mRNA, which is trans-spliced in trematodes but lacks SL sequences in four different cestode species (11), and a S. japonicum factor homologous to E. multilocularis Elp, which is obviously expressed independently of trans-splicing (39). Hence, differences in the pattern of trans-spliced factors, previously observed between trematodes and nematodes (4), can also be observed within the same phylum and concerning SLs of the same phylogenetic origin. This strongly argues against a general correlation between trans-splicing of a mRNA and the biological function of the encoded protein.

The mode of regulation of the E. multilocularis elp gene is highly remarkable. We have identified two transcripts that (i) apparently derive from alternative cis/trans-splicing at an identical splice acceptor site, (ii) only differ in a small 5' exon and in the presence or absence of a TMG cap, and (iii) obviously encode an identical protein. Although alternative cis/trans-splicing at internal introns was observed before in S. mansoni (7, 40), this was always accompanied by the formation of full-length and truncated forms of the encoded protein and, therefore, probably associated with regulatory aspects acting at the protein level. The production of an identical protein by alternative cis/trans-spliced transcripts is, to our knowledge, a novel finding and raises several questions regarding its transcriptional regulation and biological significance. By use of reporter gene constructs in C. elegans, Conrad et al. (41, 42) previously showed that introduction or removal of a splice donor site upstream of a given splice acceptor site can alter the mode of splicing of resulting mRNAs from cis to trans. This led to the hypothesis that solely the presence or absence of a 5' splice site upstream of a 3' splice site, and not the sequence context around the splice acceptor, determines the mode of splicing. Our data on the presence of two alternative elp primary transcripts, one containing the full-length 5' intron, the other a truncated version, are in perfect agreement with this hypothesis. It is, therefore, reasonable to assume that the 5' cis-spliced elp transcript derives from the longer primary transcript, whereas the 5' trans-spliced elp mRNA is determined by the truncated pre-mRNA. Why should E. multilocularis express Elp via transcripts spliced either conventionally or in trans? On the one hand, this could be a simple consequence of developmentally regulated switching between both elp promoters, leading to different pre-mRNAs for metacestode and protoscolex, or differential expression of elp as part of monocistronic and polycistronic messages, accompanied by resolving polycistrons via trans-splicing. Due to the constitutive expression pattern of conventional and trans-spliced elp mRNAs in two developmental stages and the probable expression of elp as a monocistron, we consider these scenarios unlikely. The constant relation between the conventionally and trans-spliced elp transcripts in developmental stages as different as metacestode and protoscolex indicates that both transcripts are most probably expressed at the same time within the same cell. It remains to be established whether this expression pattern is related with particular, as yet unknown functions or control mechanisms of the gene product in the echinococcal cell. Currently, experiments are performed in our laboratory to measure differential stability, translational efficiency, and intracellular location of the alternative elp transcripts. We predict that a closer analysis of the unusual splicing mode of elp mRNAs will lead to valuable insights concerning the biological significance of SL trans-splicing.

The discovery of trans-splicing in protozoans, nematodes, and trematodes has greatly facilitated the PCR-based construction of full-length cDNA libraries from these organisms (5, 37, 43-46). Particularly in the case of parasitic helminths, of which usually only limited amounts of biological material are available, the presence of identical leaders on mRNA subsets proved of high value for the identification of novel genes (5, 37, 46, 47). Based on our results, similar investigations are now possible for the two important human parasitic cestodes E. multilocularis and E. granulosus, and possibly for other cestodes such as Taenia solium or Taenia saginata. The conserved utilization of trans-splicing in all three major groups of parasitic helminths, nematodes, trematodes, and cestodes offers a potential target for the development of chemotherapeutic agents against a variety of parasitic diseases.


    ACKNOWLEDGEMENTS

We thank Kerstin Kronthaler for excellent technical assistance and Kerstin Hubert for many helpful suggestions.


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant Fr689/9-2 (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ29365-AJ29377.

Dagger To whom correspondence should be addressed. Tel.: 931-201-3936; Fax: 931-201-3445; Email: kbrehm@hygiene.uni-wuerzburg.de.

Published, JBC Papers in Press, September 5, 2000, DOI 10.1074/jbc.M006091200


    ABBREVIATIONS

The abbreviations used are: SL, spliced leader; SL-RNA, spliced leader RNA; TMG, 2,2,7-trimethylguanosine; RACE, rapid amplification of cDNA ends; RT, reverse transcription; PCR, polymerase chain reaction; nt, nucleotide(s); bp, base pair(s); kb, kilobase(s); ORF, open reading frame; snRNA, small nuclear RNA.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Nilsen, T. W. (1993) Annu. Rev. Microbiol. 47, 413-440
2. Nilsen, T. W. (1995) Mol. Biochem. Parasitol. 73, 1-6
3. Blumenthal, T. (1995) Trends Genet. 11, 132-136
4. Davis, R. E. (1996) Parasitol. Today 12, 33-40
5. Blaxter, M., and Liu, L. (1996) Int. J. Parasitol. 26, 1025-1033
6. Donelson, J. E., and Zeng, W. (1990) Parasitol. Today 6, 327-334
7. Rajkovic, A., Davis, R. E., Simonsen, J. N., and Rottman, F. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8879-8883
8. Davis, R. E., Singh, H., Botka, C., Hardwick, C., Ashraf el Meanawy, M., and Villanueva, J. (1994) J. Biol. Chem. 269, 20026-20030
9. Davis, R. E. (1997) Mol. Biochem. Parasitol. 87, 29-48
10. Ross, L. H., Freedman, J. H., and Rubin, C. S. (1995) J. Biol. Chem. 270, 22066-22075
11. Davis, R. E., and Hodgson, S. (1997) Mol. Biochem. Parasitol. 89, 25-39
12. Maroney, P. A., Denker, J. A., Darzynkiewicz, E., Laneve, R., and Nilsen, T. W. (1995) RNA (N. Y.) 1, 714-723
13. Thomas, J. D., Conrad, R. C., and Blumenthal, T. (1988) Cell 54, 533-539
14. Liou, R. F., and Blumenthal, T. (1990) Mol. Cell. Biol. 10, 1764-1768
15. Van Doren, K., and Hirsh, D. (1990) Mol. Cell. Biol. 10, 1796-1772
16. Rausch, R. L. (1995) in Echinococcus and Hydatid Disease (Thompson, R. C. A. , and Lymbery, A. J., eds) , pp. 89-134, CAB International, Wallingford, UK
17. Brehm, K., Jensen, K., Frosch, P., and Frosch, M. (1999) Mol. Biochem. Parasitol. 100, 147-152
18. Hubert, K., Cordero, E., Frosch, M., and Solomon, F. (1999) Cell Motil. Cytoskeleton 42, 178-188
19. Jura, H., Bader, A., Hartmann, M., Maschek, H. J., and Frosch, M. (1996) Infect. Immun. 64, 3484-3490
20. Frosch, P., Mühlschlegel, F., Sygulla, L., Hartmann, M., and Frosch, M. (1994) Parasitol. Res. 80, 703-705
21. Brehm, K., Kronthaler, K., Jura, H., and Frosch, M. (2000) Mol. Biochem. Parasitol. 107, 297-302
22. McManus, D. P., Knight, M., and Simpson, J. G. (1985) Mol. Biochem. Parasitol. 16, 251-266
23. Adams, D. S., Noonan, D., Burn, T. C., and Skinner, H. B. (1987) Gene 54, 93-103
24. Adams, D. S., Herrera, R. J., Luhrmann, R., and Lizardi, P. M. (1985) Biochemistry 24, 117-125
25. Diviacco, S., Norio, P, Zentilin, L., Menzo, S., Clementi, M., Biamonti, G., Riva, S., Falaschi, A., and Giacca, M. (1992) Gene 122, 313-320
26. Jayagopala Reddy, N. R., Wilkie, B. N., and Mallard, B. A. (1996) Biotechniques 21, 868-875
27. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 61-68
28. Hemmings, L., and McManus, D. P. (1991) Mol. Biochem. Parasitol. 44, 53-62
29. Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Nucleic Acids Res. 15, 8783-8798
30. Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999) J. Mol. Biol. 288, 911-940
31. El-Dabaa, E., Mei, H., El-Sayed, A., Karim, A. M., Eldeskoy, H. M., Fahim, F. A., LoVerde, P. T., and Saber, M. A. (1998) J. Parasitol. 84, 954-960
32. Kozak, M. (1991) J. Cell Biol. 115, 887-903
33. Shiwaku, K., and Donelson, J. E. (1995) Biochem. Biophys. Res. Commun. 211, 49-53
34. Carranza, S., Baguna, J., and Riutort, M. (1997) Mol. Biol. Evol. 14, 485-497
35. Keller, M., Tessier, L. H., Chan, R. L., Weil, J. H., and Imbault, P. (1992) Nucleic Acids Res. 20, 1711-1715
36. Aksoy, S., Shay, G. L., Villanueva, M. S., Bears, C. B., and Richards, F. F. (1992) Gene 113, 239-243
37. Davis, R. E., Hardwick, C., Tavernier, P., Hodgson, S., and Singh, H. (1995) J. Biol. Chem. 270, 21813-21819
38. Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyshka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596
39. Kurtis, J. D., Ramirez, B. L., Wiest, P. M., Dong, K. L., El-Meanawy, A., Petzke, M. M., Johnson, J. H., Edmison, J., Maier, R. A., and Olds, G. R. (1997) Infect. Immun. 65, 344-347
40. Hamdan, F. F., and Ribeiro, P. (1998) Parasitol. Res. 84, 839-842
41. Conrad, R., Thomas, J., Spieth, J., and Blumenthal, T. (1991) Mol. Cell. Biol. 11, 1921-1926
42. Conrad, R., Liou, R. F., and Blumenthal, T. (1993) EMBO J. 12, 1249-1255
43. Martin, S. A., Thompson, F. J., and Devaney, E. (1995) Mol. Biochem. Parasitol. 70, 241-245
44. El-Sayed, N. M. A., Alarcon, C. M., Beck, J. C., Sheffield, V. C., and Donelson, J. E. (1995) Mol. Biochem. Parasitol. 73, 75-90
45. Levick, M. P., Blackwell, J. M., Connor, V., Coulson, R. M. R., Miles, A., Smith, H. E., Wan, K. L., and Ajioka, J. W. (1996) Mol. Biochem. Parasitol. 76, 345-348
46. Blaxter, M. L., Raghavan, N., Ghosh, I., Guiliano, D., Lu, W., Williams, S. A., Slatko, B., and Scott, A. L. (1996) Mol. Biochem. Parasitol. 77, 77-93
47. Henkle-Duhrsen, K., Tawe, W., Warnecke, C., and Walter, R. D. (1995) Biochem. J. 308, 441-446

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