Abstract
Approximately 25% of Caenorhabditis elegans genes are organized as operons. Polycistronic transcripts are converted to monocistronic mRNAs by 3' cleavage/polyadenylation and 5' trans-splicing with untranslated, 5' termini of mRNAs encoded by downstream genes in operons are acceptors for > or = 7 recently discovered "novel" SLs and a classical SL (SL2). Diversity in SL exons is now partly explained by the discovery and characterization of five novel genes that encode C. elegans SL RNAs. These novel SL RNAs contain a 22- or 23-nucleotide SL followed by conserved splice donor and downstream sequences that are essential for catalysis of trans-splicing reactions. The SL3 alpha, SL4, and SL5 RNA genes are tightly clustered on chromosome III; their 114-nucleotide transcripts deliver three distinct SLs to mRNAs. The SL3 beta and SL3 gamma RNA genes are on chromosome I, but are not tightly linked. SL RNAs 3 alpha, 3 beta, and 3 gamma provide identical 5' leader exons, although their 3' sequences diverge. Transcription of SL 3-5 RNA genes appears to be driven by flanking DNA elements that are homologous with segments of promoters for the C. elegans SL2 RNA and small nuclear RNA genes. RNase protection assays demonstrated that novel SL RNAs are transcribed in vivo and accumulate in the poly(A-) RNA pool. SL3 exons are transferred to mRNAs as frequently as SL2 exons. In contrast, SL4 is appended to mRNAs 10% as frequently as SL3. The abundance of SL4 RNA increased 6-fold during postembryonic development, and the SL4 RNA gene promoter is active principally in hypodermal cells.
Highlights
Homologous with segments of promoters for the C. el- Unlike many other eukaryotes, C. elegans contains numeregans SL2 RNA and small nuclear RNA genes
Organization and Sequences of SL RNA Genes — Genomic from C. elegans — Nonclassical SLs that appear at the 5' ends of DNA inserts from recombinantphage clones designated λSLA, cDNAs encoding three distinct proteins are shown in Fig. 1. λSLB, and λSLC were char
Copy Number and Chromosomal Location of Novel SL RNA Genes - DNA fragments that account for >90% of the C. elegans genome were amplified in yeast artificial chromosomes (YACs) vectors and immobilized on a gridded filter [27]
Summary
Growth of C. elegans —The Bristol N2 strain of C. elegans was grown, synchronized, and harvested as described in previous publications [21, 22]. Digestion of λSLC with HindIII yielded a 1-kbp DNA fragment that hybridized with the SLb probe This fragment was cloned into pGEM7Z and sequenced as described below. Upon digestion with Sad, this recombinant plasmid yielded a 1.3-kbp genomic DNA fragment and linearized vector (3.0 kbp) fused with 1.0 kbp of C. elegans DNA Both species of DNA hybridized with the SLb probe. DNA inserts from plasmids that contain individual SL RNA genes and their contiguous 5' and 3' flanking sequences (see above) were used as templates to generate randomly primed, 32P-labeled probes [21]. Amplified DNAs were digested with SphI and SalI and cloned into the C. elegans expression vector pPD16.51 [33], which was cleaved with the same enzymes This places the putative SL4 RNA gene promoter upstream from a nuclear localization signal and the lacZ reporter gene. SL4 RNA:lacZ chimeric genes and the rol-6 gene, which provides a selectable marker phenotype [34], were co-injected into the gonadal syncytium of C. elegans, and stable lines of transgenic nematodes were established as described previously [35]
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