RNA processing and transcription termination in C. elegans.

Abstract

Transcription termination is an important process that allows RNA polymerase II to disengage from the DNA template after the complete synthesis of the pre-mRNA. The mechanism of Pol II termination is dependent on and tightly coupled to the recognition of a functional poly(A) site at the end of protein expressing genes. Two major models addressing the mechanism of termination have been proposed in the late eighties and experimental data in support of both models have since been presented. The anti-terminator model proposes the loss or gain of a factor that causes the polymerase to terminate upon poly(A) site recognition. In the torpedo model, the cleavage of the pre-mRNA at the poly(A) site creates an access point for an exoribonuclease. Subsequent digestion of the 3 RNA cleavage product somehow induces termination. However, a number of eukaryotes including the nematode C. elegans, have the ability to transcribe multiple genes from a single promoter resulting in polycistronic pre-mRNAs. Individual mature translatable mRNAs are generated by a process, which involves the trans-splicing of a short leader sequence at the 5 end and cleavage and polyadenylation at the 3 end of each individual gene within the polycistronic RNA. Therefore, in a polycistronic environment, several poly(A) signals are recognized that could cause the loss of polymerase associated ‘anti-termination factors and multiple cleavage reactions occur during transcription which could provide potential exoribonuclease entry points. In both cases, mechanisms must exist that prevent Pol II termination from occurring prematurely. To gain a general insight into transcription termination in the nematode we first analysed how far transcription proceeds into the 3 flanking region in five individual worm genes using RT-PCR, nuclear run on and ChIP analysis. Surprisingly and despite the fact that C. elegans contains a compact genome, we found that transcription in four of the five nematode genes proceeds almost 1kb past the poly(A) site before cleavage and subsequent termination occurs. In addition, our results further suggest that terminal intron removal and transcription termination are not necessarily coupled in nematode genes. Finally, our analysis of internal poly(A) site use in polycistronic transcripts allows us to suggest a novel mechanism as to how transcription termination may be prevented despite the recognition of multiple functional internal poly(A) sites.