[
European Worm Meeting,
2008]
Transcription termination is an important process that ensures. disengagement of RNA polymerase II (pol II) from the DNA after the. transcription of a gene. There are currently two models that describe the. molecular mechanism of this process. It is well established that the. nematode C. elegans arranges part of its genome in structures that resemble. bacterial operons and transcribe multiple genes into polycistronic. transcripts. Interestingly, the two above mentioned termination models can. be applied to explain pol II termination from yeast to man; however, they. both fail to adequately explain how premature transcription termination is. prevented in a polycistronic transcription unit at the end of each gene.. This phenomena makes C. elegans a particularly interesting organism to. study transcription termination.. A big obstacle to use C. elegans for the analysis of transcriptional. studies has so far been a lack of suitable methods. We have recently. developed several experimental protocols that allow us to analyze nascent. transcription in mixed stage C. elegans cultures. We have used these. approaches to address how 3'' end processing and transcription termination. are coupled in C. elegans. Surprisingly, in this analysis we found that,. despite the compact C. elegans genome, transcription can continue up to 0.8. kb downstream of poly(A) sites before cleavage and subsequent transcription. termination occurs. Furthermore, we have evidence that after the cleavage. at the poly(A) site transcription can continue to more than 1.5 kb. downstream of the poly(A) site. This could potentially result in several. previously unknown overlapping transcription units. It also appears that. unlike in mammals, terminal intron removal and pol II termination in the C.. elegans are not coupled.. In addition, it has so far been hard to study gene expression in C. elegans. operons because of the difficulty to detect and analyse polycistronic pre-. mRNAs. However, we have developed an experimental protocol that allows us. to readily detect and analyze polycistronic pre-mRNAs from randomly chosen. operons. Using this technique, we were able to demonstrate that several. predicted operons in C. elegans are truly transcribed as polycistronic. primary transcripts. Furthermore we show that cis-splicing at the. downstream positioned genes in the selected operons can occur prior to both. poly(A) cleavage and trans-splicing at the upstream intergenic regions.. Finally our data allows us to propose a model that describes how premature. transcription termination is prevented in C. elegans operons.
[
International Worm Meeting,
2007]
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.