Identification of transcription start sites of trans-spliced genes: Uncovering unusual operon arrangements

Abstract

In Caenorhabditis elegans, the transcripts of many genes are trans-spliced to an SL1 spliced leader, a process that removes the RNA extending from the transcription start site to the trans-splice site, thereby making it difficult to determine the position of the promoter. Here we use RT-PCR to identify promoters of trans-spliced genes. Many genes in C. elegans are organized in operons where genes are closely clustered, typically separated by only ~100 nucleotides, and transcribed by an upstream promoter. The transcripts of downstream genes are trans-spliced to an SL2 spliced leader. The polycistronic precursor RNA is processed into individual transcripts by 3' end formation and trans-splicing. Although the SL2 spliced leader does not appear to be used for other gene arrangements, there is a relatively small number of genes whose transcripts are processed by SL2 but are not close to another gene in the same orientation. Although these genes do not appear to be members of classical C. elegans operons, we investigated whether these might represent unusual operons with long spacing or a different, nonoperon mechanism for specifying SL2 trans-splicing. We show transcription of the entire region between the SL2 trans-spliced gene and the next upstream gene, sometimes several kilobases distant, suggesting that these represent exceptional operons. We also report a second type of atypical "alternative" operon, in which 3' end formation and trans-splicing by SL2 occur within an intron. In this case, the processing sometimes results in a single transcript, and sometimes in two separate mRNAs.