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Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: Trans-splicing may play an important role in the diversification of scorpion venom peptides
peptides 27 (2006) 675–681
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journal homepage: www.elsevier.com/locate/peptides
Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: Trans-splicing may play an important role in the diversification of scorpion venom peptides§ Xian-Chun Zeng 1,2, Feng Luo 2, Wen-Xin Li * State Key Laboratory of Virology, Institute of Virology, Department of Biotechnology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China
article info
abstract
Article history:
A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 50 -terminal region of
Received 30 March 2005
BmKT’ cDNA and the 30 -terminal region of an undocumented cDNA (named BmKu), was
Received in revised form
isolated from a cDNA library made from the venom gland of scorpion Buthus martensii
29 July 2005
Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-
Accepted 29 July 2005
residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot
Published on line 14 September 2005
hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse tran-
Keywords:
scription. Genomic amplifications performed with three pairs of BmKT-u gene-specific
Trans-splicing
primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single
Venom peptide
transcriptional unit. Genomic cloning for BmKT’ showed that the BmKT’ gene contains an
Evolution
intron of 509 bp inserted into the region encoding the C-terminal region of the signal
Peptide diversity
peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT’
Scorpion
revealed that the junction site of the hybrid molecule is located at the 50 -splicing site of the
Buthus martensii Karsch
intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA
Intron
that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom
Na+-channel specific toxin
peptides from venomous animals. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Scorpions are one of the most ancient groups of animals on earth. They have survived for more than 400 million years with no detectable changes in their anatomy due to their efficient and dynamic ability to produce a diverse group of neurotoxins [9]. Scorpions have developed a vast variety of venom peptides §
to block or modulate a diverse array of ion channels and other receptors in enemy and prey species. These venom peptides are 13–76 amino acid residues long. Most of them are toxins that specifically act on ion channels, including Na+, K+, CI or Ca2+ channels [6,27,28]. Although the primary structures of these toxins are highly divergent, they share a common, dense core typically formed by an a-helix and two to three b-strands,
GenBank Accession number: BmKT-u cDNA, AY055476; BmKT’ genomic sequence, AY786186. * Corresponding author. E-mail addresses: [email protected] (X.-C. Zeng), [email protected] (W.-X. Li). 1 Present address: OIIB/NIDCR, National Institutes of Health, Building 30, Room 304, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel.: +1 301 496 6060; fax: +1 301 402 1064. 2 Contribute equally to this article. 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.07.016
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cross-linked by three or four disulfide-bridged. The evolutionary conserved protein fold seen in scorpion toxins can accommodate insertions, replacements, deletions and mutations which confer on them a diverse set of specificities or new functions [13,15]. In addition, scorpion venoms have been shown to also contain low molecular weight polypeptides that do not possess disulfide-bridges [5,31,33]. The repertoire of polypeptides without disulfide bridges include antimicrobial peptides, signaling molecules involved in modulating the immune response [21,29] and bradykinin-potentiating peptides [20]. Scorpion venoms provide a rich source of biologically active peptides. However, much less is known about how peptide structural and functional diversity is genetically generated. It has become clear that venom peptide diversity occurs via gene duplication events followed by subsequent and independent gene mutations [10]. In addition, posttranslational protein modification also appears to play an important role in producing peptide diversity [33]. The protein coding sequences of most eukaryotic precursor RNAs (pre-mRNAs) are interrupted by introns which are removed by splicing. Alternative splicing, which removes introns from a pre-mRNA and selectively joins different exons to produce different mature RNAs, is an important source of protein diversity [19]. A particular type of alternative splicing, trans-splicing, may also contribute to protein diversity [17]. Trans-splicing joins exons from two independently transcribed pre-mRNAs to form a single mature transcript. Trans-splicing is considered to be one of the most important methods for producing protein diversity in invertebrates [16]. Recently, Zhu et al. isolated a novel cDNA clone (BmTXKb– BmKCT) that represents a hybrid obtained from two different mRNAs (the 50 -terminal region of BmTXKb mRNA and the 30 terminal region of BmKCT mRNA) [35]. The junction site is a 11 bp region shared in common by both BmTXKb and BmKCT cDNAs. The BmTXKb–BmKCT cDNA does not appear to have been produced by a trans-splicing event for the following reasons: (a) the junction site location and sequence is incompatible with it arising from a 50 -splice donor of the BmTXKb intron; (b) the hybrid cDNA was easily obtained in vitro through a homology-dependent template switching mechanism during the process of reverse transcription [30]. Thus, so far, no alternative- or trans-splicing event has been reported from scorpion or other poisonous animals. We believe that scorpions (and other venomous animals) are likely to successfully produce a large repertoire of peptides via several means including alternative splicing or transsplicing. In this paper, we report that trans-splicing plays a role in the scorpion venom peptide diversification. We obtained a peculiar cDNA clone, BmKT-u, from Buthus martensii Karsch that is a hybrid of the 50 -terminal region of BmKT’ cDNA (which encodes a Na+-channel-specific toxinlike peptide), and the 30 -terminal part of an undocumented cDNA (named BmKu). The junction site of the hybrid molecule is consistent with 50 -splicing site of the intron of the BmKT’ gene. Northern blotting showed that BmKT-u is a naturally occurring transcript. Genomic amplification experiments showed that BmKT-u gene does not exist in the scorpion genome as a single transcriptional unit. Since RT-PCR experiments excluded the possibility that the BmKT-u cDNA
is an artifact generated during reverse transcription, BmKT-u mRNA is likely generated by trans-splicing.
2.
Materials and methods
2.1.
cDNA library construction
Venom glands of scorpions, obtained from Henan Province, China, were cut off 2 days after extraction of their venom by electrical stimulation, and frozen immediately in liquid nitrogen. Total RNA was extracted from homogenized gland tissue using a modified single-step method. Poly (A)+mRNA was purified using a PolyATract mRNA Isolation kit (Promega). A cDNA library was constructed using the SuperScript Plasmid System (GIBCO/BRL).
2.2.
cDNA library screening
A subtractive strategy was used to isolate the clones which share a common 50 -terminal region with the cDNA encoding BmKT’, a Na+-channel specific toxin-like peptide with 66 amino acid residues from B. martensii Karsch [34], but possess different 30 -terminal regions. At first, about 5000 clones from the cDNA library were screened using a gene-specific probe that corresponds to the 50 UTR and the signal peptide region of BmKT cDNA with high stringency. Approximately 500 positive clones were screened again using a probe specific to 30 terminal sequence of BmKT’ cDNA. The negative clones were selected for sequencing.
2.3.
Sequencing of cDNA clones
Plasmid DNA was prepared using QIAGEN Plasmid Mini kit (QIAGEN GmbH, Germany). Nucleotide sequences were determined with the universal M13 forward and reverse primers from both directions by automated sequencing (ABI 377 Sequencer, Applied Biosystems) using the ABI Prism1 Big DyeTM Terminator cycle sequencing ready reaction kit according to the manufacturer’s protocol (Applied Biosystems).
2.4.
Bioinformatics analysis
Sequence similarity searches were performed using the NCBI BLAST routine (http://www.ncbi.nlm.nih.gov/blast). Sequence multiple alignments were obtained by using the Launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align) software. Sequences representing signal peptides were identified using SignalP (http://www.hcbs.dtu.dk/services) software. Predictions of secondary structural elements of the polypeptides was obtained by using a software routine at the NPS@ sever (http://npsa-pbil.ibcp.fr/).
2.5.
Northern blot hybridization
Total RNA was extracted from the venom gland of B. martensii Karsch (10 mg), separated by electrophoresis on a 2% agarose formaldehyde gel, and transferred to nylon membranes. The RNA in the membrane was hybridized with two radiolabeled BmKT-u gene-specific probes, respectively, at 65 8C in Quickhyb
peptides 27 (2006) 675–681
hybridization solution (Stratagene) following standard protocols. One probe was a DNA fragment of 125 bp which corresponds to the nucleotides from 26 to 150 of BmKT-u cDNA while the other probe was a 150 bp fragment that corresponds to the nucleotides from 74 to 223 of the cDNA. A 32P-radiolabeled probe was prepared using a random primer labeling kit (Stratagene). The hybridized membrane was washed with high stringency.
2.6. Genomic amplification and cloning of BmKT0 and BmKT-u genes High molecular weight genomic DNA was extracted from the muscles of scorpions as described previously [11]. Two or three micrograms of genomic DNA were used for PCR amplification. Three pairs of primers (primers 1 and 2, primers 3 and 4, primers 5 and 6) were used to amplify BmKT-u: primer 1 (forward), 50 -CCATAAAACGGTTCAAAATG-30 , which corresponds to the 50 UTR and the initiation codon of BmKT-u cDNA; primer 2 (reverse), 50 -CAAGTCCATCATATCTTTCC-30 , which corresponds to C-terminal part of BmKT-u peptide; primer 3 (forward), 50 -ATGAATTAT TTGGTAT TTTTTAGTTTG30 , which corresponds to N-terminal part of BmKT-u signal peptide; primer 4 (reverse), 50 -TCATCATTATCATAGTGTGG-30 , which corresponds to partial sequence of BmKT-u 30 UTR; primer 5 (forward), 50 -TGGCACTTCTTGTAATGACA-30 , which corresponds to C-terminal part of BmKT-u signal peptide; primer 6 (reverse), 50 -TACTTGAGATCCCGTCATG-30 , which corresponds to partial sequence of BmKT-u 30 UTR. One pair of primers was used to amplify the genomic sequence of BmKT’: forward primer, 50 -CTTTCCCGGAAAATTCC-30 , which corresponds to the partial sequence of BmKT’ 50 UTR; reverse primer, 50 -CAGTTATATTTAGGATACATTTC-30 , which corresponds to the partial sequence of BmKT’ 30 UTR.
2.7.
Semi-quantitative RT-PCR
Semi-quantitative RT-PCR was carried out to compare the numbers of BmKT-u first-strand cDNA molecules that were synthesized using several different reverse transcriptases or at several different reaction temperatures. Total RNA was prepared from scorpion venom gland using TRIZOL LS Reagent (GIBCO/BRL). First-strand cDNA, which was used as PCR template, was synthesized with Superscript II reverse transcriptase (RNaseH) or AMV reverse transcriptase (RNaseH+) at 37 or 45 8C. An Oligo(dT)12–18 primer was used to prime the synthesis. The primers used to amplify BmKT-u were: 50 TTCCCGGAAAATTCCATAAAACGG-30 (forward) which corresponds to first 24 nucleotides of the 50 UTR of BmKT-u cDNA and 50 -TCCTGTTCGTCAAATATACTTGAG-30 (reverse) which corresponds to a partial sequence of 30 UTR of the cDNA.
3.
Results
3.1.
Evaluation of the cDNA library
The alkaline electrophoresis analysis of 32P-labeled first strand cDNA showed that a major cluster of cDNAs which range in size from 250 to 600 nucleotides was observed (data not
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shown). This observation suggested that our cDNA library is composed of cDNA inserts that are near full-length copies of the mRNAs from which they were derived, and the cDNA library is of high quality.
3.2.
Characterization of BmKT-u cDNA
A novel full-length cDNA clone was isolated (named BmKT-u, as shown in Fig. 1). BmKT-u cDNA is 375 bp long and is composed of three regions: a 30 UTR, an open reading frame and a 50 UTR. The 30 UTR contains three AAA motifs that are thought to be involved in the regulation of mRNA stability. The initiation codon ATG is flanked by three adenines AAA. A putative polyadenylation signal (AATAAA) is found 18 nucleotides upstream from poly(A) tail. These features are similar to those of scorpion venom peptide cDNAs previously described. BmKT-u cDNA codes for a precursor of 30 amino acid residues, including a predicted signal peptide of 20 amino acid residues and a highly hydrophilic mature peptide of 10 residues that has no disulfide bridges. The signal peptide is cleaved from its precursor at a small neutral amino acid residue (Thr) and is similar to other scorpion toxin signal peptides. However, BmKT-u is the shortest scorpion venom peptide identified so far. Secondary structure prediction analysis suggests that BmKT-u possesses a coiled coile structure. A coiled coil structure was also found in other non-disulfide-bridged scorpion peptides, such as Peptide T and BmKa1 [8,31,33]. The calculated isoelectric point of BmKT-u peptide is 9.51. Similarity searches revealed that the nucleotide sequence of BmKT-u cDNA is related to that of BmKT’ gene which codes for a homologue of a Na+-channel-specific toxin, BmKT from B. martensii Karsch [34]. The 50 UTR of BmKT-u cDNA (from nucleotides 1 to 30) is identical to that of BmKT’ cDNA with the exception of one nucleotide difference at position 18 (A in BmKT-u ! G in BmKT); the nucleotide sequence from 31 to 76 which codes for the first 15 residues of the signal peptide is identical to the corresponding region of the BmKT’ cDNA. However, the remaining sequence of BmKT-u cDNA showed no any similarity with the corresponding part of the BmKT’ cDNA. These findings strongly suggest that the BmKT-u transcript derives from a recombination event between BmKT’ and an unknown gene in genomic or mRNA level, or just an artificial hybrid molecule generated during reverse transcription through homology-dependent template switching.
3.3. The BmKT-u gene doesn’t exist in the scorpion genome as a single transcriptional unit Based on the cDNA sequence of BmKT-u, we designed three pairs of primers to amplify its genomic sequence. Because of the possibility of genomic polymorphism, different scorpions may possess sequences that present a template mismatch to our primers; therefore we used a mixture of genomic DNAs isolated from a number of scorpions to serve as templates. We also collected scorpions of the same species from several sources to obtain our genomic DNA preparation. In every experiment, we set up two PCR reactions to amplify the genomic sequences of BmKK2 and BmTX2 as positive control reactions. Using these procedures we failed to obtain any PCR product for all amplifications of BmKT-u (data not shown);
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Fig. 1 – Alignment of the sequence of BmKT-u cDNA with the genomic and the cDNA sequence of BmKT’. Deduced amino acid sequences of BmKT-u and BmKT’ precursors are given below or above their corresponding nucleotide sequences, respectively. The nucleotide sequence is numbered from the first base pair. The amino acid residue sequence is numbered starting from the N-terminal amino acid residue of the peptide; the first residue of putative mature peptide is numbered +1, whereas the signal peptide is numbered minus numbers. Signal peptide is underlined; a potential polyadenylation signal (AATAAA) is indicated by box.
however, all the positive control reactions yielded PCR products. Furthermore, we amplified the fragment from nucleotides 1 to 76, and the fragment from 77 to 375, respectively, showing that the two fragments do exist in the genome. Because the longest intron of all scorpion venom peptide genes described so far is no more than 3.0 kb long, the PCR method is sufficient to amplify any scorpion peptide gene. Our data convincingly showed that the BmKT-u DNA
sequence doesn’t exist in the scorpion genome as a single transcriptional unit. Therefore, it is not likely that the BmKT-u cDNA derives from a genomic gene recombination event.
3.4.
BmKT-u cDNA is actually generated from a transcript
Northern blotting was performed to determine whether BmKT-u mRNA is expressed in the venom gland of the
peptides 27 (2006) 675–681
Fig. 2 – Northern blotting analysis of BmKT-u transcript from venom gland of scorpion B. martensii Karsh. (a) Hybridized using probe A which is a DNA fragment of 125 bp that corresponds to the nucleotides from 26 to 150 of BmKT-u cDNA; (b) hybridized using probe B which is a fragment of 150 bp that corresponds to the nucleotides from 74 to 223 of BmKT-u cDNA.
scorpion. Hybridization with probe A that includes partial sequences of both BmKT’ and BmKu cDNAs yielded two different bands (about 600 and 450 bp) (Fig. 2a). The 450 bp band is the expected size of BmKT-u mRNA, but the expected size of BmKT’ mRNA is also about 450 bp. So the band of 450 bp may represent either the BmKT-u or BmKT’ mRNA. The band of 600 bp likely represents BmKu mRNA. If the hybridization was performed with probe B which only includes a fragment of BmKu cDNA, similar results were also observed (Fig. 2b). The data clearly demonstrates that the band of 450 bp represents BmKT-u transcript, and the band of 600 bp represent BmKu transcript. Therefore, BmKT-u cDNA is generated from a transcript.
3.5. Is the BmKT-u cDNA an artifact generated in the reverse transcription process by template-switching? It is well documented that RNase H RT (reverse transcriptase lacking RNase H activity) may produce much lower homologydependent template-switching frequency than RNase H+ RT. Luo and Taylor reported that a M-MuLV-reverse transcriptase lacking RNase H function switched about 27 times less frequently than the enzyme with RNase activity [18]. Other studies showed that RNase H is a requirement for the homology-dependent strand transfer, and RNase H-deficient reverse transcriptase completely lost template-switching ability [24]. Moreover, the AMV reverse transcriptase template switching is temperature-dependent. Therefore, we conducted semi-quantitative RT-PCR to amplify BmKT-u under
Fig. 3 – Semi-quantitative RT-PCR analysis to compare the synthesis frequency of the first-strand cDNA of BmKT-u under different conditions. Lane 1, template cDNA was synthesized using Superscript II reverse transcriptase (RNsseHS) at 37 8C; Lane 2, cDNA synthesized using AMV reverse transcriptase (RNase H+) at 37 8C; Lane 3, cDNA synthesized using AMV reverse transcriptase (RNase H+) at 45 8C. PCR amplification was carried out for 30 cycles of 30 s at 95 8C, 30 s at 58 8C, 30 s at 72 8C.
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different conditions to test whether RNase H activity or temperature affects the amount of PCR products. If a negative result is obtained then it suggests that the BmKT-u transcript occurs naturally. Our data showed that under different conditions (RNase H+, 37 8C; RNase H, 37 8C; RNase H+, 45 8C), the amount of PCR products are not significantly different (0.7:1:0.8) (Fig. 3). Thus, BmKT-u cDNA is really transcribed from a naturally occurring mRNA.
3.6. Characterization of genomic sequence of BmKT’ and its alignment with BmKT-u cDNA Finally, we tried to clone the genomic sequence of a mammal toxin that has a 50 -region in common with BmKT-u cDNA. Because there are a large number of BmKT’ toxin homologues in the scorpion venom, locating a genomic clone that exactly matches the 50 -terminal region of BmKT-u cDNA is not a simple task. After sequencing five clones we obtained the genomic sequence of BmKT’. As shown in Fig. 1, the BmKT’ genomic gene contains two exons interrupted by an intron of 509 bp, which inserts in the 5 position of the signal peptide. The split site is within the codon for Gly, between the first base G and second base G. The intron begins with GT and ends with AG, which is consistent with all the previously described intron junctions. The sequences of the 50 -splice donor and 30 splice acceptor are 50 -Gjgtaag and tacagjG, respectively, which are in concert with the consensus found in introns of other scorpion toxin genes. The A + T content of the intron is very high (77%). These data show the genomic organization of BmKT’ is highly similar to genes that code for other Na+channel-specific toxins from scorpions [7,12,25]. The alignment of the sequence of BmKT-u cDNA with the genomic sequence of BmKT’ is shown in Fig. 1. The comparison suggests that the BmKT-u cDNA is a chimera formed by the combination of the exon 1 of BmKT’ gene, and an exon of an unknown gene (BmKu). The junction site for the chimeric gene is exactly located at the 50 -splicing site of the intron of BmKT’ gene. The right side of the junction point, jGC, is consistent with 30 -splicing site of the intron of BmTx3A gene from scorpion B. martensii Karsch [14]. These results suggest that the BmKT-u mRNA derives from a trans-splicing event.
4.
Discussion
In order to understand the evolution of scorpion venom peptides and their genes, it is worthwhile to gain insight into the evolution of toxins from other venomous animals. Studies of snake venom have shown that a diverse array of peptides displaying a variety of biochemical and pharmacological functions exist. Evolutionary analysis of the genes of several snake toxin multigene families suggests that snake toxins have evolved structurally and functionally via gene duplication and mutation events, and positive selective pressures to retain functional toxins [10,15,36]. Also, surprisingly, the three different regions of a Conus peptide precursor, including signal sequence, pro-peptide and mature peptide have diverged at remarkably different rates [4,23]. The three regions are separated by introns. This striking segment-specific divergence rate of Conus peptide pre cursors suggests a role
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for introns in evolution. Olivera et al. [23] suggested that unconventional mechanisms, perhaps similar to recombination events are required to explain this evolutionary phenomenon. However, no evidence has been found to support this hypothesis so far. Molecular evolution studies also suggest that each scorpion toxin superfamily evolved from a common ancestral gene via duplication events followed by positive Darwinian selection [10]. However, this evolutionary model can not explain all the relationship among different toxin superfamilies. Some toxins from dramatically different scorpion toxin families share some common structural or functional domains. For example, BmKbpp, an antimicrobial peptide, possesses a unique Cterminal region which is highly similar to the sequence of Peptide K 12, a bradykinin-potentiating peptide from scorpion [32]. Such phenomenon is likely to arise from a recombination event. In this paper we first report that a recombination event in mRNA level, called trans-splicing actually occurs during the splicing of animal toxin pre-mRNAs. In vitro studies showed that trans-splicing reaction requires either a 50 -splicing site or exonic splicing enhancers [1,3]. Trans-splicing was first discovered in trypanosomes and later found in chordates. However, this type of trans-splicing, called spliced leaderaddition trans-splicing, only joins 50 -terminal non-coding exons into other mRNAs, and is not a source of protein diversity [22]. The best characterized functional protein that apparently originated via trans-splicing is encoded by the Drosophila mod gene [26]. Some evidences also revealed that trans-splicing occurs in mammalian cells [2]. It is not clear whether trans-splicing commonly occurs among eukaryotes. Our data convincingly showed that trans-splicing may play a significant role in the diversification of scorpion venom peptides. Because the introns of scorpion venom peptide genes are significantly polymorphous, demonstrating variations in length (from 18 to 2500 bp), position of exon interruption (inserted in signal peptide, in mature peptide or in 50 UTR) and number (1 or 2), trans-splicing may significantly cause venom peptide diversification. Transsplicing may also play a critical role in the diversification of other animal venom peptides.
Acknowledgements Authors wish to thank Professor Richard Hahin for his critical reading and Mr. Qing-Cong Zeng for his help in typing the manuscript.
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journal homepage: www.elsevier.com/locate/peptides
Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: Trans-splicing may play an important role in the diversification of scorpion venom peptides§ Xian-Chun Zeng 1,2, Feng Luo 2, Wen-Xin Li * State Key Laboratory of Virology, Institute of Virology, Department of Biotechnology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China
article info
abstract
Article history:
A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 50 -terminal region of
Received 30 March 2005
BmKT’ cDNA and the 30 -terminal region of an undocumented cDNA (named BmKu), was
Received in revised form
isolated from a cDNA library made from the venom gland of scorpion Buthus martensii
29 July 2005
Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-
Accepted 29 July 2005
residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot
Published on line 14 September 2005
hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse tran-
Keywords:
scription. Genomic amplifications performed with three pairs of BmKT-u gene-specific
Trans-splicing
primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single
Venom peptide
transcriptional unit. Genomic cloning for BmKT’ showed that the BmKT’ gene contains an
Evolution
intron of 509 bp inserted into the region encoding the C-terminal region of the signal
Peptide diversity
peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT’
Scorpion
revealed that the junction site of the hybrid molecule is located at the 50 -splicing site of the
Buthus martensii Karsch
intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA
Intron
that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom
Na+-channel specific toxin
peptides from venomous animals. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Scorpions are one of the most ancient groups of animals on earth. They have survived for more than 400 million years with no detectable changes in their anatomy due to their efficient and dynamic ability to produce a diverse group of neurotoxins [9]. Scorpions have developed a vast variety of venom peptides §
to block or modulate a diverse array of ion channels and other receptors in enemy and prey species. These venom peptides are 13–76 amino acid residues long. Most of them are toxins that specifically act on ion channels, including Na+, K+, CI or Ca2+ channels [6,27,28]. Although the primary structures of these toxins are highly divergent, they share a common, dense core typically formed by an a-helix and two to three b-strands,
GenBank Accession number: BmKT-u cDNA, AY055476; BmKT’ genomic sequence, AY786186. * Corresponding author. E-mail addresses: [email protected] (X.-C. Zeng), [email protected] (W.-X. Li). 1 Present address: OIIB/NIDCR, National Institutes of Health, Building 30, Room 304, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel.: +1 301 496 6060; fax: +1 301 402 1064. 2 Contribute equally to this article. 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.07.016
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cross-linked by three or four disulfide-bridged. The evolutionary conserved protein fold seen in scorpion toxins can accommodate insertions, replacements, deletions and mutations which confer on them a diverse set of specificities or new functions [13,15]. In addition, scorpion venoms have been shown to also contain low molecular weight polypeptides that do not possess disulfide-bridges [5,31,33]. The repertoire of polypeptides without disulfide bridges include antimicrobial peptides, signaling molecules involved in modulating the immune response [21,29] and bradykinin-potentiating peptides [20]. Scorpion venoms provide a rich source of biologically active peptides. However, much less is known about how peptide structural and functional diversity is genetically generated. It has become clear that venom peptide diversity occurs via gene duplication events followed by subsequent and independent gene mutations [10]. In addition, posttranslational protein modification also appears to play an important role in producing peptide diversity [33]. The protein coding sequences of most eukaryotic precursor RNAs (pre-mRNAs) are interrupted by introns which are removed by splicing. Alternative splicing, which removes introns from a pre-mRNA and selectively joins different exons to produce different mature RNAs, is an important source of protein diversity [19]. A particular type of alternative splicing, trans-splicing, may also contribute to protein diversity [17]. Trans-splicing joins exons from two independently transcribed pre-mRNAs to form a single mature transcript. Trans-splicing is considered to be one of the most important methods for producing protein diversity in invertebrates [16]. Recently, Zhu et al. isolated a novel cDNA clone (BmTXKb– BmKCT) that represents a hybrid obtained from two different mRNAs (the 50 -terminal region of BmTXKb mRNA and the 30 terminal region of BmKCT mRNA) [35]. The junction site is a 11 bp region shared in common by both BmTXKb and BmKCT cDNAs. The BmTXKb–BmKCT cDNA does not appear to have been produced by a trans-splicing event for the following reasons: (a) the junction site location and sequence is incompatible with it arising from a 50 -splice donor of the BmTXKb intron; (b) the hybrid cDNA was easily obtained in vitro through a homology-dependent template switching mechanism during the process of reverse transcription [30]. Thus, so far, no alternative- or trans-splicing event has been reported from scorpion or other poisonous animals. We believe that scorpions (and other venomous animals) are likely to successfully produce a large repertoire of peptides via several means including alternative splicing or transsplicing. In this paper, we report that trans-splicing plays a role in the scorpion venom peptide diversification. We obtained a peculiar cDNA clone, BmKT-u, from Buthus martensii Karsch that is a hybrid of the 50 -terminal region of BmKT’ cDNA (which encodes a Na+-channel-specific toxinlike peptide), and the 30 -terminal part of an undocumented cDNA (named BmKu). The junction site of the hybrid molecule is consistent with 50 -splicing site of the intron of the BmKT’ gene. Northern blotting showed that BmKT-u is a naturally occurring transcript. Genomic amplification experiments showed that BmKT-u gene does not exist in the scorpion genome as a single transcriptional unit. Since RT-PCR experiments excluded the possibility that the BmKT-u cDNA
is an artifact generated during reverse transcription, BmKT-u mRNA is likely generated by trans-splicing.
2.
Materials and methods
2.1.
cDNA library construction
Venom glands of scorpions, obtained from Henan Province, China, were cut off 2 days after extraction of their venom by electrical stimulation, and frozen immediately in liquid nitrogen. Total RNA was extracted from homogenized gland tissue using a modified single-step method. Poly (A)+mRNA was purified using a PolyATract mRNA Isolation kit (Promega). A cDNA library was constructed using the SuperScript Plasmid System (GIBCO/BRL).
2.2.
cDNA library screening
A subtractive strategy was used to isolate the clones which share a common 50 -terminal region with the cDNA encoding BmKT’, a Na+-channel specific toxin-like peptide with 66 amino acid residues from B. martensii Karsch [34], but possess different 30 -terminal regions. At first, about 5000 clones from the cDNA library were screened using a gene-specific probe that corresponds to the 50 UTR and the signal peptide region of BmKT cDNA with high stringency. Approximately 500 positive clones were screened again using a probe specific to 30 terminal sequence of BmKT’ cDNA. The negative clones were selected for sequencing.
2.3.
Sequencing of cDNA clones
Plasmid DNA was prepared using QIAGEN Plasmid Mini kit (QIAGEN GmbH, Germany). Nucleotide sequences were determined with the universal M13 forward and reverse primers from both directions by automated sequencing (ABI 377 Sequencer, Applied Biosystems) using the ABI Prism1 Big DyeTM Terminator cycle sequencing ready reaction kit according to the manufacturer’s protocol (Applied Biosystems).
2.4.
Bioinformatics analysis
Sequence similarity searches were performed using the NCBI BLAST routine (http://www.ncbi.nlm.nih.gov/blast). Sequence multiple alignments were obtained by using the Launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align) software. Sequences representing signal peptides were identified using SignalP (http://www.hcbs.dtu.dk/services) software. Predictions of secondary structural elements of the polypeptides was obtained by using a software routine at the NPS@ sever (http://npsa-pbil.ibcp.fr/).
2.5.
Northern blot hybridization
Total RNA was extracted from the venom gland of B. martensii Karsch (10 mg), separated by electrophoresis on a 2% agarose formaldehyde gel, and transferred to nylon membranes. The RNA in the membrane was hybridized with two radiolabeled BmKT-u gene-specific probes, respectively, at 65 8C in Quickhyb
peptides 27 (2006) 675–681
hybridization solution (Stratagene) following standard protocols. One probe was a DNA fragment of 125 bp which corresponds to the nucleotides from 26 to 150 of BmKT-u cDNA while the other probe was a 150 bp fragment that corresponds to the nucleotides from 74 to 223 of the cDNA. A 32P-radiolabeled probe was prepared using a random primer labeling kit (Stratagene). The hybridized membrane was washed with high stringency.
2.6. Genomic amplification and cloning of BmKT0 and BmKT-u genes High molecular weight genomic DNA was extracted from the muscles of scorpions as described previously [11]. Two or three micrograms of genomic DNA were used for PCR amplification. Three pairs of primers (primers 1 and 2, primers 3 and 4, primers 5 and 6) were used to amplify BmKT-u: primer 1 (forward), 50 -CCATAAAACGGTTCAAAATG-30 , which corresponds to the 50 UTR and the initiation codon of BmKT-u cDNA; primer 2 (reverse), 50 -CAAGTCCATCATATCTTTCC-30 , which corresponds to C-terminal part of BmKT-u peptide; primer 3 (forward), 50 -ATGAATTAT TTGGTAT TTTTTAGTTTG30 , which corresponds to N-terminal part of BmKT-u signal peptide; primer 4 (reverse), 50 -TCATCATTATCATAGTGTGG-30 , which corresponds to partial sequence of BmKT-u 30 UTR; primer 5 (forward), 50 -TGGCACTTCTTGTAATGACA-30 , which corresponds to C-terminal part of BmKT-u signal peptide; primer 6 (reverse), 50 -TACTTGAGATCCCGTCATG-30 , which corresponds to partial sequence of BmKT-u 30 UTR. One pair of primers was used to amplify the genomic sequence of BmKT’: forward primer, 50 -CTTTCCCGGAAAATTCC-30 , which corresponds to the partial sequence of BmKT’ 50 UTR; reverse primer, 50 -CAGTTATATTTAGGATACATTTC-30 , which corresponds to the partial sequence of BmKT’ 30 UTR.
2.7.
Semi-quantitative RT-PCR
Semi-quantitative RT-PCR was carried out to compare the numbers of BmKT-u first-strand cDNA molecules that were synthesized using several different reverse transcriptases or at several different reaction temperatures. Total RNA was prepared from scorpion venom gland using TRIZOL LS Reagent (GIBCO/BRL). First-strand cDNA, which was used as PCR template, was synthesized with Superscript II reverse transcriptase (RNaseH) or AMV reverse transcriptase (RNaseH+) at 37 or 45 8C. An Oligo(dT)12–18 primer was used to prime the synthesis. The primers used to amplify BmKT-u were: 50 TTCCCGGAAAATTCCATAAAACGG-30 (forward) which corresponds to first 24 nucleotides of the 50 UTR of BmKT-u cDNA and 50 -TCCTGTTCGTCAAATATACTTGAG-30 (reverse) which corresponds to a partial sequence of 30 UTR of the cDNA.
3.
Results
3.1.
Evaluation of the cDNA library
The alkaline electrophoresis analysis of 32P-labeled first strand cDNA showed that a major cluster of cDNAs which range in size from 250 to 600 nucleotides was observed (data not
677
shown). This observation suggested that our cDNA library is composed of cDNA inserts that are near full-length copies of the mRNAs from which they were derived, and the cDNA library is of high quality.
3.2.
Characterization of BmKT-u cDNA
A novel full-length cDNA clone was isolated (named BmKT-u, as shown in Fig. 1). BmKT-u cDNA is 375 bp long and is composed of three regions: a 30 UTR, an open reading frame and a 50 UTR. The 30 UTR contains three AAA motifs that are thought to be involved in the regulation of mRNA stability. The initiation codon ATG is flanked by three adenines AAA. A putative polyadenylation signal (AATAAA) is found 18 nucleotides upstream from poly(A) tail. These features are similar to those of scorpion venom peptide cDNAs previously described. BmKT-u cDNA codes for a precursor of 30 amino acid residues, including a predicted signal peptide of 20 amino acid residues and a highly hydrophilic mature peptide of 10 residues that has no disulfide bridges. The signal peptide is cleaved from its precursor at a small neutral amino acid residue (Thr) and is similar to other scorpion toxin signal peptides. However, BmKT-u is the shortest scorpion venom peptide identified so far. Secondary structure prediction analysis suggests that BmKT-u possesses a coiled coile structure. A coiled coil structure was also found in other non-disulfide-bridged scorpion peptides, such as Peptide T and BmKa1 [8,31,33]. The calculated isoelectric point of BmKT-u peptide is 9.51. Similarity searches revealed that the nucleotide sequence of BmKT-u cDNA is related to that of BmKT’ gene which codes for a homologue of a Na+-channel-specific toxin, BmKT from B. martensii Karsch [34]. The 50 UTR of BmKT-u cDNA (from nucleotides 1 to 30) is identical to that of BmKT’ cDNA with the exception of one nucleotide difference at position 18 (A in BmKT-u ! G in BmKT); the nucleotide sequence from 31 to 76 which codes for the first 15 residues of the signal peptide is identical to the corresponding region of the BmKT’ cDNA. However, the remaining sequence of BmKT-u cDNA showed no any similarity with the corresponding part of the BmKT’ cDNA. These findings strongly suggest that the BmKT-u transcript derives from a recombination event between BmKT’ and an unknown gene in genomic or mRNA level, or just an artificial hybrid molecule generated during reverse transcription through homology-dependent template switching.
3.3. The BmKT-u gene doesn’t exist in the scorpion genome as a single transcriptional unit Based on the cDNA sequence of BmKT-u, we designed three pairs of primers to amplify its genomic sequence. Because of the possibility of genomic polymorphism, different scorpions may possess sequences that present a template mismatch to our primers; therefore we used a mixture of genomic DNAs isolated from a number of scorpions to serve as templates. We also collected scorpions of the same species from several sources to obtain our genomic DNA preparation. In every experiment, we set up two PCR reactions to amplify the genomic sequences of BmKK2 and BmTX2 as positive control reactions. Using these procedures we failed to obtain any PCR product for all amplifications of BmKT-u (data not shown);
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Fig. 1 – Alignment of the sequence of BmKT-u cDNA with the genomic and the cDNA sequence of BmKT’. Deduced amino acid sequences of BmKT-u and BmKT’ precursors are given below or above their corresponding nucleotide sequences, respectively. The nucleotide sequence is numbered from the first base pair. The amino acid residue sequence is numbered starting from the N-terminal amino acid residue of the peptide; the first residue of putative mature peptide is numbered +1, whereas the signal peptide is numbered minus numbers. Signal peptide is underlined; a potential polyadenylation signal (AATAAA) is indicated by box.
however, all the positive control reactions yielded PCR products. Furthermore, we amplified the fragment from nucleotides 1 to 76, and the fragment from 77 to 375, respectively, showing that the two fragments do exist in the genome. Because the longest intron of all scorpion venom peptide genes described so far is no more than 3.0 kb long, the PCR method is sufficient to amplify any scorpion peptide gene. Our data convincingly showed that the BmKT-u DNA
sequence doesn’t exist in the scorpion genome as a single transcriptional unit. Therefore, it is not likely that the BmKT-u cDNA derives from a genomic gene recombination event.
3.4.
BmKT-u cDNA is actually generated from a transcript
Northern blotting was performed to determine whether BmKT-u mRNA is expressed in the venom gland of the
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Fig. 2 – Northern blotting analysis of BmKT-u transcript from venom gland of scorpion B. martensii Karsh. (a) Hybridized using probe A which is a DNA fragment of 125 bp that corresponds to the nucleotides from 26 to 150 of BmKT-u cDNA; (b) hybridized using probe B which is a fragment of 150 bp that corresponds to the nucleotides from 74 to 223 of BmKT-u cDNA.
scorpion. Hybridization with probe A that includes partial sequences of both BmKT’ and BmKu cDNAs yielded two different bands (about 600 and 450 bp) (Fig. 2a). The 450 bp band is the expected size of BmKT-u mRNA, but the expected size of BmKT’ mRNA is also about 450 bp. So the band of 450 bp may represent either the BmKT-u or BmKT’ mRNA. The band of 600 bp likely represents BmKu mRNA. If the hybridization was performed with probe B which only includes a fragment of BmKu cDNA, similar results were also observed (Fig. 2b). The data clearly demonstrates that the band of 450 bp represents BmKT-u transcript, and the band of 600 bp represent BmKu transcript. Therefore, BmKT-u cDNA is generated from a transcript.
3.5. Is the BmKT-u cDNA an artifact generated in the reverse transcription process by template-switching? It is well documented that RNase H RT (reverse transcriptase lacking RNase H activity) may produce much lower homologydependent template-switching frequency than RNase H+ RT. Luo and Taylor reported that a M-MuLV-reverse transcriptase lacking RNase H function switched about 27 times less frequently than the enzyme with RNase activity [18]. Other studies showed that RNase H is a requirement for the homology-dependent strand transfer, and RNase H-deficient reverse transcriptase completely lost template-switching ability [24]. Moreover, the AMV reverse transcriptase template switching is temperature-dependent. Therefore, we conducted semi-quantitative RT-PCR to amplify BmKT-u under
Fig. 3 – Semi-quantitative RT-PCR analysis to compare the synthesis frequency of the first-strand cDNA of BmKT-u under different conditions. Lane 1, template cDNA was synthesized using Superscript II reverse transcriptase (RNsseHS) at 37 8C; Lane 2, cDNA synthesized using AMV reverse transcriptase (RNase H+) at 37 8C; Lane 3, cDNA synthesized using AMV reverse transcriptase (RNase H+) at 45 8C. PCR amplification was carried out for 30 cycles of 30 s at 95 8C, 30 s at 58 8C, 30 s at 72 8C.
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different conditions to test whether RNase H activity or temperature affects the amount of PCR products. If a negative result is obtained then it suggests that the BmKT-u transcript occurs naturally. Our data showed that under different conditions (RNase H+, 37 8C; RNase H, 37 8C; RNase H+, 45 8C), the amount of PCR products are not significantly different (0.7:1:0.8) (Fig. 3). Thus, BmKT-u cDNA is really transcribed from a naturally occurring mRNA.
3.6. Characterization of genomic sequence of BmKT’ and its alignment with BmKT-u cDNA Finally, we tried to clone the genomic sequence of a mammal toxin that has a 50 -region in common with BmKT-u cDNA. Because there are a large number of BmKT’ toxin homologues in the scorpion venom, locating a genomic clone that exactly matches the 50 -terminal region of BmKT-u cDNA is not a simple task. After sequencing five clones we obtained the genomic sequence of BmKT’. As shown in Fig. 1, the BmKT’ genomic gene contains two exons interrupted by an intron of 509 bp, which inserts in the 5 position of the signal peptide. The split site is within the codon for Gly, between the first base G and second base G. The intron begins with GT and ends with AG, which is consistent with all the previously described intron junctions. The sequences of the 50 -splice donor and 30 splice acceptor are 50 -Gjgtaag and tacagjG, respectively, which are in concert with the consensus found in introns of other scorpion toxin genes. The A + T content of the intron is very high (77%). These data show the genomic organization of BmKT’ is highly similar to genes that code for other Na+channel-specific toxins from scorpions [7,12,25]. The alignment of the sequence of BmKT-u cDNA with the genomic sequence of BmKT’ is shown in Fig. 1. The comparison suggests that the BmKT-u cDNA is a chimera formed by the combination of the exon 1 of BmKT’ gene, and an exon of an unknown gene (BmKu). The junction site for the chimeric gene is exactly located at the 50 -splicing site of the intron of BmKT’ gene. The right side of the junction point, jGC, is consistent with 30 -splicing site of the intron of BmTx3A gene from scorpion B. martensii Karsch [14]. These results suggest that the BmKT-u mRNA derives from a trans-splicing event.
4.
Discussion
In order to understand the evolution of scorpion venom peptides and their genes, it is worthwhile to gain insight into the evolution of toxins from other venomous animals. Studies of snake venom have shown that a diverse array of peptides displaying a variety of biochemical and pharmacological functions exist. Evolutionary analysis of the genes of several snake toxin multigene families suggests that snake toxins have evolved structurally and functionally via gene duplication and mutation events, and positive selective pressures to retain functional toxins [10,15,36]. Also, surprisingly, the three different regions of a Conus peptide precursor, including signal sequence, pro-peptide and mature peptide have diverged at remarkably different rates [4,23]. The three regions are separated by introns. This striking segment-specific divergence rate of Conus peptide pre cursors suggests a role
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for introns in evolution. Olivera et al. [23] suggested that unconventional mechanisms, perhaps similar to recombination events are required to explain this evolutionary phenomenon. However, no evidence has been found to support this hypothesis so far. Molecular evolution studies also suggest that each scorpion toxin superfamily evolved from a common ancestral gene via duplication events followed by positive Darwinian selection [10]. However, this evolutionary model can not explain all the relationship among different toxin superfamilies. Some toxins from dramatically different scorpion toxin families share some common structural or functional domains. For example, BmKbpp, an antimicrobial peptide, possesses a unique Cterminal region which is highly similar to the sequence of Peptide K 12, a bradykinin-potentiating peptide from scorpion [32]. Such phenomenon is likely to arise from a recombination event. In this paper we first report that a recombination event in mRNA level, called trans-splicing actually occurs during the splicing of animal toxin pre-mRNAs. In vitro studies showed that trans-splicing reaction requires either a 50 -splicing site or exonic splicing enhancers [1,3]. Trans-splicing was first discovered in trypanosomes and later found in chordates. However, this type of trans-splicing, called spliced leaderaddition trans-splicing, only joins 50 -terminal non-coding exons into other mRNAs, and is not a source of protein diversity [22]. The best characterized functional protein that apparently originated via trans-splicing is encoded by the Drosophila mod gene [26]. Some evidences also revealed that trans-splicing occurs in mammalian cells [2]. It is not clear whether trans-splicing commonly occurs among eukaryotes. Our data convincingly showed that trans-splicing may play a significant role in the diversification of scorpion venom peptides. Because the introns of scorpion venom peptide genes are significantly polymorphous, demonstrating variations in length (from 18 to 2500 bp), position of exon interruption (inserted in signal peptide, in mature peptide or in 50 UTR) and number (1 or 2), trans-splicing may significantly cause venom peptide diversification. Transsplicing may also play a critical role in the diversification of other animal venom peptides.
Acknowledgements Authors wish to thank Professor Richard Hahin for his critical reading and Mr. Qing-Cong Zeng for his help in typing the manuscript.
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