Although eukaryotic genes are usually transcribed individually, at least a few Caenorhabditis elegans genes appear to be transcribed polycistronically in clusters resembling bacterial operons. The spliced leader SL2 (ref. 2) is specific for trans-splicing to downstream genes in these operons. In addition, many C. elegans pre-mRNAs are trans-spliced to SL1 (ref. 3) near the 5' ends of pre-mRNAs. Because operons have not previously been found in higher eukaryotes, we have investigated how widespread they are in the C. elegans genome. We identified gene clusters using the extensive data generated by the genome project and tested seven for trans-splicing specificity. All were found to fit expectations for polycistronic transcription. In addition, we surveyed reported C. elegans genes for trans-splicing specificity. Both methods indicate that the pre-mRNAs of about 70% of C. elegans genes are trans-spliced and as many as a quarter are transcribed in operons.

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.

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We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1␤-D-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or ␣-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3-end processing is mediated by transcriptional coupling.In eukaryotic cells, pol II synthesizes pre-mRNA that is processed in the nucleus to become mature mRNA and is then exported to the cytoplasm. Capping, splicing, and 3Ј-end processing are interdependent and often occur cotranscriptionally on the nascent transcript at the DNA template (2, 5, 32). Processing can also occur posttranscriptionally after release from the site of transcription (3, 37). The carboxy-terminal domain (CTD) of the largest subunit of pol II (Rpb1) provides an important link between transcription and processing by acting as a landing pad that binds directly to processing factors and localizes them to the site of transcription (4,7,13,14,23,31). In mammalian cells, pol II lacking the CTD produces transcripts that are not efficiently capped, spliced, or cleaved at poly(A) sites (24,25). Furthermore, in vitro the CTD can enhance capping, splicing, and poly(A) site cleavage uncoupled from transcription (15-17, 33, 40, 42, 43). These results suggest that the CTD of pol II that is not transcriptionally engaged can act as an allosteric activator of pre-mRNA processing reactions. Although the CTD is important for pre-mRNA processing, pol II transcription is by no means essential. RNA precursors can be processed in vitro and, in some cases, in vivo in the absence of transcription. Introns appearing early in the premRNA of Chironomus BR1 and BR3 genes are predominantly spliced at the site of transcription, whereas introns close to the 3Ј end are spliced after the transcript has been released (3, 37). It is also possible that cleavage and polyadenylation occurs posttranscriptionally, because cleavage frequently does not precede termination (29). It is not known if pol II that is not transcriptionally engaged can facilitate pre-mRNA processing in vivo after release from the site of transcription.During transcription, the CTD undergoes extensive phosphorylation and dephosphorylation on Ser2 and Ser5 residues of the heptad repeats (YSPTSPS). CTD hyperphosphorylation by CDK7 and CDK9 is associated with the transition from initiation to elongation (19,21). Protein kinase inhibitors, including 5,6-dichloro-1␤-D-ribofuranosyl...