mRNA Trans-Splicing in the Human Parasitic Cestode ... · 9). In this study we have investigated gene expression mechanisms in the cestode Echinococcus multilocularis , the so-called

mRNA Trans-Splicing in the Human Parasitic Cestode Echinococcus

multilocularis

Klaus Brehm*, Katharina Jensen, and Matthias Frosch

Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Straße 2,

97080 Würzburg, Germany

* To whom correspondence should be addressed: Tel: +931-201-3936; Fax: +931-201-

3445; Email: [email protected]

Running title:

Trans-splicing in the cestode Echinococcus multilocularis §

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 5, 2000 as Manuscript M006091200 by guest on June 18, 2020

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SUMMARY

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) which is

obviously spliced in trans 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 which 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 ERM protein family, is expressed via two alternative transcripts, spliced

either in cis or in 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

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Trans-splicing is a mechanism of mRNA processing which 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 which 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 in cis, usually at introns within the coding region, and in trans, at the 5’ end (1-5). A further

difference is that in metazoans not all mRNAs are trans-spliced. In C. 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 which 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 which 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 S. 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 MMG-

cap (13-15).

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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 E. 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 ERM-family of proteins (17, 18), is expressed via two alternatively spliced transcripts of which one

contains an echinococcal spliced leader at its 5’ end. We have identified several echinococcal mRNAs which are

processed by trans-splicing and provide a detailed characterization of the SL encoding gene and the SL-RNA.

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EXPERIMENTAL PROCEDURES

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 (19)

and protoscoleces were isolated as described in (21). Chromosomal DNA and total RNA was 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 (Quiagen, Hilden, Germany) was used

according to the manufacturers 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

manufacturers instructions.

5’- and 3’-RACE- 5’-RACE was performed using oligo-dG-tailing of ss-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 polyA-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 polyA-tail according to a previously described

protocol (23). The SL RNA 3’ end was subsequently determined by sequencing of obtained PCR products.

Preparation of polyA+ RNA and TMG-immunoprecipitation- For the isolation of polyA+ and polyA- RNA

fractions the Oligotex mRNA purification -KIT (Quiagen, Hilden, Germany) was used according to the

manufacturers instructions. After repeated application of total RNA to oligo-dT columns, both the polyA+ and

the polyA- 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, Schwalbach, Germany).

PCR and RT-PCR amplification- For standard PCR amplifications, the Eurogentec (Seraign, Belgium)

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GoldStar Taq-system was used. For the amplification of long products the Advantage 2 PCR Kit (Clontech,

Palo Alto, CA, USA) was employed according to the manufacturers instructions. In RT-PCR reactions, mRNA

was reverse transcribed using Omniscript reverse transcriptase (Quiagen, Hilden, Germany) and subsequently

either PCR amplified as described above or using the One Step PCR Kit (Quiagen, Hilden, Germany). Detection

of pre-mRNA was performed using the sensitive Titan One Step RT-PCR System of Boehringer (Mannheim,

Germany). 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 polyA-tail. The

resulting ss-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, Palo Alto, Ca, USA) 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 which 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

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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., 94 °C, 1 min., 57 °C, 1 min., 72 °C and using the primer combinations EC5 x E10-6 for elp

transcript A as well as ET5 x 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.

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RESULTS

Identification of alternatively spliced elp transcripts - We have previously characterized the chromosomal elp-

locus and investigated elp transcription by 5’-RACE (17). One transcript, herein termed transcript A, was

identified which 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 further 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 polyA 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.

< Figure 1 suggested >

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 Meriones 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 specifical 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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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) which 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. 3A/B) which 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 which 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 observable.


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 which 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“) which were specific for the SL but which

were oriented in opposite directions. After PCR amplification from chromosomal E. multilocularis DNA as a

template, a 1513 bp fragment was obtained, cloned in E. 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

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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-loop 2

and 3, and the typical location of the SL within the first stem-loop are present.

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 as for 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, 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 which shared 87 % homology with the E. multilocularis

repeat. Two nucleotide substitutions were observed for the SL-RNA of which one 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 F. 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.


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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 which 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 ss-cDNA with primers specific for the polyA-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 3 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 revealed significant database matches with already known proteins. Six were homologous to

proteins with unknown functions. The remaining 11 are listed in table 1. Concerning the putative biological

function of the encoded proteins, no common patterns were identified although 3 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 1).

< Table 1 suggested >

With its 3’ end the echinococcal SL provides trans-spliced mRNAs with an AUG codon which, 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 1). In 3 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

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translation initiation. For 2 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 which 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 which were located close to the 5’ intron’s splice-donor

(„10-24“) and splice-acceptor sites („10-19“), respectively. Interestingly, with the combination 10-6 x 10-19,

a PCR product was obtained which, 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 x 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 further 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 x 10-29 still revealed a completely spliced fragment for the introns I -

III while the combination 10-6 x 10-31 gave no product (Fig. 5, Fig. 1).


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

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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.

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DISCUSSION

Several evidences, provided in this study, are clearly indicative of SL trans-splicing in E. multilocularis: (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 5S 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 β-tubulin encoding transcripts of E. multilocularis (21)

which are exclusively expressed without spliced leader. Based on a preliminary analysis of 150 randomly

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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 which can

differentiate between TMG and MMG containing transcripts (38). It will be worthwhile to investigate whether

alternative eIF4E 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. heptica (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. Like 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 which are expressed via

trans-splicing. Although the echinococcal UbCRBP and FBPA 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 4 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 which (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

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(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).

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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 T. 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.

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FOOTNOTES:

§ This work was supported by grant Fr689/9-2 from the Deutsche Forschungsgemeinschaft (to M.F.). The costs

of publication of this article were defrayed in part by the payment of page charges. This 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/EBI Data Bank with

accession number(s) AJ29365 – AJ29377

1 The abbreviations used are: SL, spliced leader; SL-RNA, spliced leader RNA; snRNA,

small nuclear RNA; TMG, 2,2,7-trimethylguanosine; MMG, 7-methylguanosine; ERM,

ezrin/radixin/moesin; RACE, rapid amplification of cDNA ends; RT, reverse transcription;

PCR, polymerase chain reaction; ss, single stranded; nt, nucleotide(s); bp, basepair(s); kb,

kilobase(s); UbCRBP, ubiquinol-cytochrome C-reductase binding protein; FBPA, fructose-

bisphosphate-aldolase.

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LEGENDS

Table 1. Trans-spliced mRNAs of E. multilocularis. A cDNA library for trans-spliced

mRNAs of isolate H95 was constructed as indicated in experimental procedures. The inserts

of 11 recombinant clones which yielded significant BLAST scores were entirely sequenced

and are listed. Indicated are for each clone: the cDNA size (in bp; excluding the polyA-tail

and the 36 nt SL sequence), the length of the predicted ORF (in amino acids), the homology

(in % identical amino acids) of the deduced amino acid sequence to the best matching amino

acid sequence in databases, the predicted identification according to homology, and the

accession numbers of best matching proteins together with their origin. Further 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 Em 1431, 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.

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

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determined by 5’ RACE is shown. Exonic sequences are represented by capitals, intronic and

non-transcribed regions by small letters. Size and location of elp exon I, exon II, and the 5’

intron are indicated by lines and small arrows (<__>) 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 (---à) 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 upper case letters.

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, 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 1x106 (lane1), 5x105 (lane 2), 2,5x105 (lane3), 1,2x105

(lane4), 6x104 (lane 5), and 3x104 (lane 6) molecules of the competitor which was constructed as described in

experimental procedures. For competitive PCR the primer combinations 10-6 x EC5, specific for the exon I-

containing transcript A (A) and 10-6 x 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, the lower to the cDNA. Equimolarity

between competitor and cDNA was reached at ~ 1.2x105 molecules for transcript A and ~ 2.5x105 molecules

for transcript B in the protoscolex fraction. For the metacestode fraction, the respective values were ~ 2.5x105

for transcript A and ~ 5x105 for transcript B. Thus, a constant relation of about 1:2 between transcripts A and B

was measurable in both developmental stages.

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Fig. 3. Northern blot analysis of transcripts containing the SL. A) Analysis of polyA+ and polyA- fractions. E.

multilocularis was cultivated in Meriones unguiculatus and parasite RNA was isolated. 10 µg total RNA (lanes

1, 4) and equivalent portions of polyA- (lane 2) and polyA+ 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, 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 immunopreciptation 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.

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 (11). Nucleotides identical to that of

the E. multilocularis SL RNA are marked by dots (.) and dashes (-) represent gaps inserted to maximize the

alignment. Further 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. Indicated are: the splice site by an arrow; the SL in bold letters; the

trimethylguanosine-cap (TMG) 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.

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 (striped boxes).

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Oligonucleotide primers used for mapping the transcriptional start point are indicated by small arrows. B) PCR

and RT-PCR analysis. 2 µg total RNA of the E. multilocularis isolates H95 (lanes 1, 4, 7) and K188 (lanes 2, 5,

8) were RT-PCR amplified in a 30 µl volume employing a sensitive one step RT-PCR system (Titan,

Boehringer, Mannheim, Germany) and using the primer combinations 10-31 x 10-6 (lanes 1, 2), 10-29 x 10-6

(lanes 4, 5), and 10-19 x 10-6 (lanes 7, 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 x

10-6; lane 6: 10-29 x 10-6; lane 9: 10-19 x 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, 8). No products were obtained for 10-31 x 10-6 although this primer combination gave a clear positive signal

on chromosomal DNA.

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Figure 1

Chromosome <___________Exon I ______________><_donor 5 ’ Intron ___________________________________àgttgcttGCCCGTGGTTGGGCTCCCGTTGATTGCAGTTT//gtgcgt..<171bp>..ttcacttctcgtgaacagtagcctccttgtttgag. Primer „ 10-31“ --------------------à Primer „10-29“ -----------------à

à__5 ’ Intron acceptor _><____________Exon II __________________..<230bp>..cttatattttag//ACTAAAACCATGTTGAAGAGGAGTAAGAA........ M L K R S K N........

Transcript A 5’-GCCCGUGGUUGGGCUCCCGUUGAUUGCAGUUU-ACUAAAACCAUGUUGAAGAGGAGUAAGAA... Exon I M L K R S K N

Transcript B 5’-CACCGUUAAUCGGUCCUUACCUUGCAGUUUUGUAUG-ACUAAAACCAUGUUGAAGAGGAGUAAGAA... spliced leader M L K R S K N


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Figure 4

A). . . . .Em CACCGTTAATCGGTCCTTACCTTGCAGTTT--TGTATGGTGAGTATCGAT 48Eg ..........................A........... ............ 48Fh A...T.-..CG.T..TC.G..C..T.TA..AG..C... ..A..A....T. 48Sm A.....-C.CG.T.TTAC-T....TGA...GT..C... ..A..A.C..TC 48

. . . . .Em GCAGC-TGAGGCTGTGCCTACGAGCTGAC---CCAGTATTTGGCTGGTCC 94Eg .......C.......................................... 94Fh .G.C.A.CG.T.CAAA..C.TT.TT..G.TAG..TCC...CT..A.AGG. 98Sm .-.C.AA..AT.GAA.TT--TTCTT..G.AGC..TAC.CAC----..GG. 91

hom. to E. mult.- . length: SL SL-snRNAEm TTCGAGGGCC 104 100% 100%Eg .......... 104 97% 98%Fh .AA..AT-CC 107 55% 34%Sm .A 93 50% 30%

B)

G U U C-G G-C G-C A-U A C G-C U G G U-A U-A C-G C-G C-G G-C U-G U U U A-U G-CU UGUA GGUGAG AUCGAUGC-GACCCAGTATTTGGCUGG-C |||| |||||| ||||||U ACGU CCAUUC UGGCUA G U C A U U GCCAC-*TMG-cap


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Table 1

Clone cDNA ORF- %ID Identification Accession- Organism 5‘endSize length No.

Em1222 1053 223 59 RAS-related Protein RAB 4-A SP:P56371 M. musculus ..ATG/GAAAAATCAGGTCTCTAAAATGGAA.. M E

Em1211 598 130 50 Ubiquinol Cyt.C Reductase SP:O01369 F. hepatica ..ATG/GAGGTCTTCTTAACACTGCTCTGAGAATGACC..Complex subunit (UbCRBP) M T

Em4231 308 83 85 snRNA-associated PRF:2514316E H. sapiens ..ATG/CATGCAA..Sm-like protein M Q

Em1342 1285 311 79 Translation-initiation- SP:P38919 H. sapiens ..ATG/GTCAATGGCG..Factor 4A M A

Em2222 1301 363 73 Fructose-Bisphosphate- SP:ALF_SCHMA S. mansoni ..ATG/AAAAATGTCT..Aldolase (FBPA) M S

Em3332 2101 641 52 Spliceosome-associated SP:S114_HUMAN H. sapiens ..ATG/TTTAACTGATCATGACT..Protein SAP 114 M T

Em4212 590 188 75 U-snRNP-associated GP:AF016371_1 H. sapiens ..ATG/CAGAGAATGGCA..Cyclophilin M A

Em2342 1394 433 41 Mannose-6-Phosphate- SP:MANA_HUMANH. sapiens ..ATG/ATCATGCTT..Isomerase M L

Em2313 372 82 67 Enhancer of Rudimentary SP:ERH_HUMAN H. sapiens ..ATG/ATGTCG..Homolog M S

Em1433 2173 335 30 Cyclin H SP:CYCH_HUMAN H. sapiens ..ATG/GCTTAC.. M A Y

Em1431 432 127 67 High-Mobility-Group-like SP:NHPX_HUMAN H. sapiens ..ATG/GCTGAA..Protein 2 Homolog M A E

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Klaus Brehm, Katharina Jensen and Matthias FroschmRNA trans-splicing in the human parasitic cestode Echinococcus multilocularis

published online September 5, 2000J. Biol. Chem.

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mRNA Trans-Splicing in the Human Parasitic Cestode ... · 9). In this study we have investigated gene expression mechanisms in the cestode Echinococcus multilocularis , the so-called