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Trans-Spliced Leader Addition to Mrnas in a Cnidarian

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Trans-Spliced Leader Addition to Mrnas in a Cnidarian Trans-spliced leader addition to mRNAs in a cnidarian Nicholas A. Stover and Robert E. Steele* Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine, CA 92697-1700 Edited by Stanley N. Cohen, Stanford University School of Medicine, Stanford, CA, and approved March 8, 2001 (received for review February 3, 2000) A search of databases with the sequence from the 5؅ untranslated Materials and Methods region of a Hydra cDNA clone encoding a receptor protein-tyrosine Database Searches. Database searches were carried out by using kinase revealed that a number of Hydra cDNAs contain one of two the BLAST server at the National Center of Biotechnology .(different sequences at their 5؅ ends. This finding suggested the Information (16 possibility that mRNAs in Hydra receive leader sequences by trans-splicing. This hypothesis was confirmed by the finding that Isolation of Hydra DNA and RNA. Genomic DNA was isolated from the leader sequences are transcribed as parts of small RNAs the Zurich strain of Hydra vulgaris essentially as described by encoded by genes located in the 5S rRNA clusters of Hydra. The two Davis et al. (17). Total H. vulgaris RNA was extracted with hot spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucle- acidic phenol (18). otides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Rapid Amplification of cDNA Ends (RACE). 5Ј RACE for the HTK32 Both of the SL RNAs are bound by antibody against trimethyl- cDNA was performed essentially according to Frohman (19). guanosine, suggesting that they contain a trimethylguanosine cap. PolyAϩ RNA was isolated from adult H. vulgaris polyps by using The predicted secondary structures of the Hydra SL RNAs show an RNeasy Midi Kit (Qiagen, Chatsworth, CA) and an Oligotex significant differences from the structures predicted for the SLs of mRNA Mini Kit (Qiagen). Reverse transcription was performed by other organisms. Messenger RNAs have been identified that can using a gene-specific primer (5Ј-TTGTAGCTCTTACATTAC-3Ј). receive either SL-A or -B, although the impact of the two different The resulting first-strand cDNA was polydA tailed with terminal SLs on the function of the mRNA is unknown. The presence and transferase (Boehringer Mannheim) before the first round of PCR. features of SL addition in the phylum Cnidaria raise interesting The initial amplification reaction was carried out by using a mixture questions regarding the evolution of this process. of three primers as described by Frohman (19). The sequences of the primers are as follows: 5Ј-CAATAACTCTATATTTACC-3Ј; 5Ј-AAGGATCCGTCGACATCG-3Ј;5Ј-AAGGATCCGTCGA- n members of a small number of phyla, leader sequences obtained CATCGATAATACGACTCACTATAGGGATTTTTTTT- from a small nuclear RNA, the spliced leader (SL) RNA, are I TTTTTTTT-3Ј. Amplification conditions were as described by attached to the 5Ј ends of mRNAs. To date, SL addition has been Frohman (19). The second round of amplification was for 40 cycles identified in three metazoan phyla (Nematoda, Platyhelminthes, at a final annealing temperature of 44°C by using the following and Chordata) (1–4) and in one unicellular eukaryotic phylum primer pair: 5Ј-GTATAAACACCAGCATC-3Ј;5Ј-ATCGATA- (Sarcomastigophora) (3, 5–7). No evidence of SL addition has been ATACGACTCAC-3Ј. The 390-bp fragment obtained from the detected in any intensively studied plants, fungi, insects, echino- second amplification reaction was purified by agarose gel electro- derms, or vertebrates. The phylogenetic distribution of SL addition phoresis, extracted from the gel by using the Qiaex II Gel Extraction (Fig. 1) is surprising and has made it difficult to discern the Kit (Qiagen), and cloned into the pGEM-T EZ cloning vector evolutionary history of this process. However, SL additions in (Promega). unicellular eukaryotes and metazoans do share several features. For 3Ј RACE of SL-A and -B, polyA tails were added to the total Many SL RNAs have a highly conserved structure that includes RNA sample by using cloned yeast polyA polymerase (United three stem–loops, and all of the SL RNAs have a binding site for States Biochemical). The polyadenylation reaction was carried out the Sm protein (8). In a number of uni- and multicellular species, according to the manufacturer’s protocol. First-strand cDNA was copies of the SL RNA genes are found to be repeated within the synthesized from the pool of polyadenylated RNA by using AMV 5S rDNA cluster (9). For all of the genes in trypanosomes, for a reverse transcriptase (Boehringer Mannheim) and was primed Ј portion of nematode genes, and possibly in some flatworm genes, from an oligonucleotide containing a dT16 sequence at its 3 end SL addition serves to separate polycistronic primary transcripts into (5Ј-AAGGATCCGTCGACATCGATAATACGACTCACT- individual mRNAs (10–12). SL addition is thus an important ATAGGGATTTTTTTTTTTTTTTT-3Ј). Two rounds of touch- feature of both genome organization and gene expression in these down PCR (20) were performed to obtain the 3Ј ends of the SL-A phyla. The conservation of these features suggests the possibility and -B RNAs. For SL-A, the first round of amplification was carried that SL addition originated in unicellular eukaryotes and was out for 40 cycles at a final annealing temperature of 38°C by using retained at least into the ancestor of modern protostomes and the following pair of primers: 5Ј-GGTAGGTACCATAACAGTT- deuterostomes but was lost after the divergence of many metazoan TAC-3Ј;5Ј-AAGGATCCGTCGACATCG-3Ј. The second round phyla. However, given the apparently small number and diverse of amplification was carried out for 35 cycles at a final annealing phylogenetic positions of metazoan phyla that carry out SL addi- temperature of 49°C by using the following pair of primers: tion, multiple independent origins for this process must also be 5Ј-TCTCTTTACGATTTTCGGG-3Ј;5Ј-ATCGATAATAC- considered. Early diverging metazoan phyla have been examined for the presence of SL addition (13), but so far no cases have been reported. Here we demonstrate the presence of SLs on mRNAs in This paper was submitted directly (Track II) to the PNAS office. Hydra, a member of the early-diverging metazoan phylum Cnidaria. Abbreviations: SL, spliced leader; RACE, rapid amplification of cDNA ends; TMG, trimethylguanosine. Previous studies of Hydra genes had revealed that they undergo Data deposition: The sequences reported in this paper have been deposited in the GenBank typical cis-splicing (14, 15), with the introns in some Hydra genes database (accession nos. AF287010, AF217320, AF123442, and AF286166). being located at sites identical to those in homologous genes in *To whom reprint requests should be addressed. E-mail: [email protected]. vertebrates (14). The presence and features of SL addition in The publication costs of this article were defrayed in part by page charge payment. This EVOLUTION Cnidaria provide additional data bearing on the puzzling evolu- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. tionary history of SL addition in metazoans. §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.101049998 PNAS ͉ May 8, 2001 ͉ vol. 98 ͉ no. 10 ͉ 5693–5698 Downloaded by guest on October 1, 2021 mide. After capillary transfer to a Genescreen II nylon membrane (DuPont), the RNA was crosslinked to the membrane by using a UV Stratalinker 2400 (Stratagene). Probes labeled with [␣-32P]dATP were made by extending a primer annealed to a synthetic oligonucleotide template by using Klenow DNA polymer- ase (Promega) in Prime-It II dATP reaction buffer (Stratagene). For the SL-A intron probe, the template was 5Ј-GTAGGCAT- CAATAAATTTTGACGCAAATTTTCGAATTTTCGGGG- TTTCGGTAGTGGGTAAA-3Ј, and the primer was 5Ј-CCCAC- TACCGAAACCCCGAA-3Ј. For the SL-B exon probe, the tem- plate was 5Ј-ACGGAAAAAAACACATACTGAAACTTTT- TAGTCCCTGTGTAATAAG-3Ј, and the primer was 5Ј- CTTATTACACAGGGACTAAAAAG-3Ј. Hybridizations were carried out in an aqueous buffer [1 M NaCl, 100 mM Tris⅐HCl, pH 8.0͞0.1% BSA͞0.1% polyvinylpyrrolidone͞0.1% Ficoll͞0.05% so- dium pyrophosphate͞0.1% SDS͞0.1 mM disodium EDTA͞50 ␮g/ml heparin͞100 ␮g/ml torula yeast RNA (Sigma)͞500 ␮g/ml herring sperm DNA (Sigma)] for 18 h at room temperature. The blots were washed at room temperature twice in high salt buffer (1 M NaCl͞0.1 M Tris, pH 8.0͞0.1 mM disodium EDTA), then in 2ϫ standard saline phosphate͞EDTA [0.18 M NaCl͞10 mM phos- phate, pH 7.4͞1 mM EDTA (26)]. Hybridization was detected by exposure of the filters to x-ray film with intensifying screens at Ϫ70°C. For identification of RNAs containing trimethylguanosine ␮ Fig. 1. Phylogenetic distribution of spliced leader addition to mRNAs. (TMG) caps, 30 g of total RNA was immunoprecipitated by Phylogenetic relationships of only those taxa in which spliced leader addition using an anti-TMG antibody͞agarose conjugate [Oncogene is known to be present or likely to be absent are shown. The phylogenetic Research Products (Cambridge, MA)]. Both the bound and relationships between the taxa are based on multiple molecular studies unbound fractions were phenol͞chloroform extracted and pre- (42–44). (A) A version of the tree in which spliced leader addition arose in a cipitated in 75% ethanol. Electrophoresis, transfer, hybridiza- unicellular eukaryote and was subsequently lost from the various taxa indi- tion, and washing were all performed as described above. A cated in gray. Minuses indicate points at which loss occurred. (B) A version of probe that recognized 5S rRNA was made with the template the tree in which spliced leader addition arose independently in the various 5Ј -CCTACGACCATACCACGGTGAACACACCCGTT- phyla indicated in black. ϩ, points at which origin of spliced leader addition Ј Ј occurred. CTCGTCCGATCACGGAAG-3 and the primer 5 -CTTC- CGTGATCGGACGAGAAC-3Ј by using the same procedure as described above for preparation of SL probes. GACTCAC-3Ј. The 101-bp fragment obtained from the second amplification reaction was gel-purified and cloned into pGEM-T DNA Sequencing.
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