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Operons and SL2 Trans-Splicing Exist in Nematodes Outside the Genus Caenorhabditis (Polycistronic Transcription͞caenorhabditis Elegans͞splice Leader͞sl RNA)

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Operons and SL2 Trans-Splicing Exist in Nematodes Outside the Genus Caenorhabditis (Polycistronic Transcription͞caenorhabditis Elegans͞splice Leader͞sl RNA) Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9751–9756, September 1997 Evolution Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis (polycistronic transcriptionyCaenorhabditis elegansysplice leaderySL RNA) DONALD EVANS*, DIEGO ZORIO*, MARGARET MACMORRIS*‡,CARLOS E. WINTER†,KRISTI LEA*, AND THOMAS BLUMENTHAL*‡§ *Department of Biology, Indiana University, Bloomington, IN 47405; and †Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas–University of Sao Paulo, 05508-900, Brazil Communicated by Norman R. Pace, University of California, Berkeley, CA, June 3, 1997 (received for review February 26, 1997) ABSTRACT The genomes of most eukaryotes are com- other nematodes (ref. 7; reviewed in refs. 8 and 9), in Euglena posed of genes arranged on the chromosomes without regard (10), and in flatworms (11, 12). In contrast to trypanosomes, to function, with each gene transcribed from a promoter at its in which only trans-splicing is present, the genes in the other 5* end. However, the genome of the free-living nematode organisms also contain cis-spliced introns. It was presumed Caenorhabditis elegans contains numerous polycistronic clus- that these genes were monocistronic and arranged randomly ters similar to bacterial operons in which the genes are on the chromosomes as in other eukaryotes. However, it was transcribed sequentially from a single promoter at the 5* end recently discovered that the free-living nematode C. elegans of the cluster. The resulting polycistronic pre-mRNAs are does have polycistronic transcription units, as in trypanosomes, processed into monocistronic mRNAs by conventional 3* end and it uses a special small nuclear ribonucleoprotein, the SL2 formation, cleavage, and polyadenylation, accompanied by small nuclear ribonucleoprotein, to process the downstream trans-splicing with a specialized spliced leader (SL), SL2. To genes in the polycistronic pre-mRNAs into monocistronic determine whether this mode of gene organization and ex- mRNAs (13, 14). pression, apparently unique among the animals, occurs in Trans-splicing in nematodes involves the donation of a 22 other species, we have investigated genes in a distantly related nucleotide spliced leader (SL), including a trimethylguanosine free-living rhabditid nematode in the genus Dolichorhabditis cap, from a small nuclear RNA to the 59 ends of pre-mRNAs (strain CEW1). We have identified both SL1 and SL2 RNAs in (15–20). While the exact function of the SL is unknown, it has this species. In addition, we have sequenced a Dolichorhabditis recently been shown to increase translation efficiency of those genomic region containing a gene cluster with all of the mRNAs containing it at their 59 ends in Ascaris lumbricoides characteristics of the C. elegans operons. We show that the in vitro extracts (21). downstream gene is trans-spliced to SL2. We also present C. elegans has two different SLs, each 22 nt long. The first evidence that suggests that these two genes are also clustered to be discovered, SL1 (7), is trans-spliced onto the 59 ends of in the C. elegans and Caenorhabditis briggsae genomes. Thus, it about half of C. elegans mRNAs (14). The signal for SL1 appears that the arrangement of genes in operons pre-dates trans-splicing is the presence of a short sequence, called an the divergence of the genus Caenorhabditis from the other outron, at the 59 end of the pre-mRNA (22). The outron is genera in the family Rhabditidae, and may be more wide- simply an AU-rich sequence lacking a functional 59 splice site, spread than is currently appreciated. followed by a 39 splice site (23, 24). The second SL to be discovered, SL2 (25), was shown to be In bacteria and archaea, the genomes are primarily organized reserved for splicing to downstream genes in polycistronic in arrays of genes whose products have related functions. These pre-mRNAs derived from cotranscribed gene clusters, or gene clusters, called operons, are cotranscribed from an operons (13). The resulting polycistronic pre-mRNAs are upstream promoter and the resulting polycistronic mRNA is processed into monocistronic units by cleavage and polyade- translated by ribosomes initiating at or near the 59 end of the nylation and by trans-splicing as they are synthesized. Approx- RNA. These operons serve to efficiently coregulate proteins imately 25% of C. elegans genes are arranged in operons (14). that function together. In contrast, eukaryotes have genomes The genes in these clusters are oriented in the same 59 to 39 composed of genes arranged apparently at random, with each direction and are separated by '100 bp of intercistronic DNA, transcribed by a promoter at its 59 end. However, in a group or in rarer cases 300–400 bp (26). In every case investigated, of primitive eukaryotic protozoa, the trypanosomes, genes are the first gene in the cluster is either trans-spliced to SL1 or is transcribed polycistronically (1–3). In this case, the polycis- not trans-spliced, whereas the downstream genes are trans- tronic pre-mRNA is processed by 39 end formation and spliced, either exclusively to SL2 or to a mixture of SL2 and SL1 trans-splicing to create conventional eukaryotic monocistronic (13, 14). Since SL2 has only been found trans-spliced to mRNAs. The trans-splicing reaction that creates the 59 ends of downstream genes in operons, we interpret the presence of SL2 the mRNAs is related to the cis-splicing of higher eukaryotes; on the 59 end of an mRNA as evidence of an operon (27). it proceeds through a 29–59 branched intermediate, the splice Although C. elegans operons may not always serve to sites have the same consensus sequences, and it is catalyzed by coregulate genes whose products function together, as in some of the same small nuclear ribonucleoprotein particles (4, bacteria, there are a few clear examples of operons that serve 5). this purpose. For example the deg-3 operon contains two Trans-splicing was first discovered in trypanosomatids (4, 6), and later shown to occur also in Caenorhabditis elegans and Abbreviations: SL, spliced leader; RT, reverse transcription. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge deposited in the GenBank database (accession nos. U90830–U90835). ‡Present address: Department of Biochemistry and Molecular Genet- payment. This article must therefore be hereby marked ‘‘advertisement’’ in ics, University of Colorado Health Sciences Center, 4200 East 9th accordance with 18 U.S.C. §1734 solely to indicate this fact. Avenue, Denver, CO 80262. © 1997 by The National Academy of Sciences 0027-8424y97y949751-6$2.00y0 §To whom reprint requests should be sent at the present address. PNAS is available online at http:yywww.pnas.org. e-mail: [email protected]. 9751 9752 Evolution: Evans et al. Proc. Natl. Acad. Sci. USA 94 (1997) subunits of the same acetylcholine receptor (ref. 28; M. Treinin hybridized with either a Dolichorhabditis rpl-29 RT-PCR prod- and M. Chalfie, personal communication), and the lin-15 uct or a C. elegans rpp-1 cDNA PCR product. operon encodes two unrelated proteins whose products col- Southern Blots. Genomic DNA isolated from Dolichorhab- laborate in the cell signaling pathway resulting in vulva for- ditis, C. elegans, and C. briggsae was digested with restriction mation (29, 30). endonucleases, separated electrophoretically on a 1% agarose It is not yet known how widespread SL2 and operons are in gel, and transferred to Hybond-N. This blot was then probed the nematode phylum. SL1 has been detected in all nematode with a 283-bp Dolichorhabditis rpl-29 PCR product (oligos: species studied with virtually no sequence variation (reviewed L27B659-1, 59-GCACCCTGGAGGACGTG-39 and L27- in ref. 9). In contrast, SL2 has been found only in Caenorhab- B659-4, 59-GCCTTCTCAGCGCTGACC-39), which was la- ditis species (25). Comparison of C. elegans with Caenorhabditis beled as above. The low stringency hybridization conditions briggsae, two species that have been estimated to have been were 53 SSC, 23 Denhardt’s solution, 0.05 M NaPO4 (pH 6.5), separated for 25–50 million years (31), shows perfect conser- 0.1% SDS, and 0.2 mgyml salmon sperm DNA, incubated at vation of SL2 and in several cases, the same operons found in 48°C. The wash conditions were 23 SSC at room temperature the C. elegans genome are present in the genome of C. briggsae and 13 SSC at 50°C. The annealed probe was removed from (32–34). the blot (confirmed by autoradiographic exposure) and then In this paper we report, to the best of our knowledge, the the blot was hybridized with the C. elegans rpp-1 cDNA PCR first example of an operon and of SL2 trans-splicing in a product used to select the Dolichorhabditis clone. Conditions nematode outside of the genus Caenorhabditis: the rhabditid were the same, except that the rpp-1 probe was hybridized at Dolichorhabditis (CEW1). The isolate CEW1 is an undescribed 50°C. sibling species of Rhabditis (Oscheius) tipulae, a member of the Cloning Dolichorhabditis SL1 Genes. The Dolichorhabditis Dolichura Group (35). Placement of CEW1 and R. tipulae in a genomic library was screened with a 32P-kinased oligonucle- genus is actively under investigation (L. Carta, personal com- otide composed of the first 20 nt of the C. elegans SL1 spliced munication), but CEW1 is provisionally called Dolichorhabdi- leader (SL1–20: 59-GGTTTAATTACCCAAGTTTG-39). tis. Although Dolichorhabditis is a small free-living nematode, Plaques that hybridized to this probe were isolated. At least it is much more evolutionarily distant from C. elegans than is two different clones were isolated based on digestion with C. briggsae. This conclusion is based on the sequence of SL1 multiple restriction endonucleases. Fragments that hybridized and SL2 RNAs, as well as a comparison of the vit-6 genes from to the probe were subcloned and sequenced as above.
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