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Biology Articles » Molecular Biology » RNA polymerase II and the integration of nuclear events » Transcription termination and RNA processing: the final link

Transcription termination and RNA processing: the final link
- RNA polymerase II and the integration of nuclear events

Transcription termination and RNA processing: the final link

As we mentioned at the beginning of this review, evidence has existed for over a decade suggesting that a functional polyadenylation signal is required for RNA polymerase II to terminate transcription (for review, see Proudfoot 1989). These experiments, which employed transient transfection assays with plasmids containing mutated poly(A) signals and nuclear run-on analysis of transcription, led to two models: In one, the polymerase is modified in some way as it passes the polyadenylation signal, causing it to become less processive and more likely to terminate. In the other, the act of 3' cleavage is directly signaled to elongating RNAP II, by the action of a 5'-to-3' exonuclease that rapidly degrades the downstream product of the cleavage reaction, and this then causes the polymerase to become termination prone. Although it still remains unclear which, if either, of these models are correct, a number of different approaches have strengthened the polyadenylation-termination link, and at least placed some limitations on the possible mechanism.

Given that mutations in cis-acting 3' processing signals prevent termination, an interesting question has been whether mutations in trans-acting polyadenylation factors would have similar effects. This was studied by Birse et al. (1998), who examined termination (by nuclear run-on) in yeast cells harboring mutations in different biochemically characterized polyadenylation factors. Intriguingly, cells with mutations in factors implicated in the cleavage step were defective in termination at the nonpermissive temperature, but those in which factors thought to be involved only in the second step were affected (e.g., poly(A) polymerase, or PAP) showed no termination defects. These findings provide both genetic evidence linking 3'-end formation and transcription termination and also support for the idea that RNA cleavage is required, consistent with the second model above. However, the demonstration in mammalian systems that certain factors can associate with RNAP II (although not PAP; McCracken et al. 1997b) weakens somewhat this second conclusion, as it's possible that the mutations alter in some way these interactions so that required changes in the RNAP II complex, and hence termination, do not occur.

Two additional studies, one using modified run-on assays with transfected mammalian cells (Dye and Proudfoot 1999), the other analyzing by PCR nascent transcripts from the heavily transcribed Balbani ring 1 gene isolated from salivary glands of the diptern Chironomous tentans (Bauren et al. 1998), provided additional, and surprisingly similar, insights into termination. Consistent with previous work, both studies provided evidence for termination (i.e., the lack of run-on or PCR signals) ~1000 bp downstream of the polyadenylation signal. However, in each case the large majority of nascent RNAs detected right up to the apparent termination site were uncleaved. This suggests that perhaps 3' cleavage is not essential for termination. However, another possibility is that processing and termination are actually temporally coupled. Perhaps there is a lag in processing until RNAP II encounters a pause site, which activates 3' cleavage, and this in turn signals the polymerase to terminate and the complex to dissociate (Fig. 4). These results are consistent with the requirement of RNAP II and specifically the CTD in 3' processing (McCracken et al. 1997b; Hirose and Manley 1998) and with the demonstration that a pause site can enhance polyadenylation in a coupled processing/transcription reaction (Yonaha and Proudfoot 1999). Intriguingly, both of these studies also implicated processing, or at least recognition, of the 3' most intron in subsequent termination. This likely reflects the enhancement of polyadenylation by splicing of the upstream intron (Niwa et al. 1990), which in turn activates termination.

The above studies all analyzed termination somewhat indirectly, by assaying for the presence or absence of nascent RNAs. It is thus reassuring that a study examining the terminating polymerases more directly reached very similar conclusions (Osheim et al. 1999). These authors visualized by electron microscopy (EM) transcribing polymerases on templates isolated from microinjected Xenopus laevis oocytes. As expected, they observed termination occurring downstream of polyadenylation signals. Significantly, a functional poly(A) signal was required for termination, and the strength of the signal was directly proportional to the efficiency of termination. Consistent with the experiments just described, a majority of the transcripts approaching the termination site were uncleaved, consistent with either a lack of a cleavage requirement for termination or a temporal linkage of the two processes. Interestingly, the nascent transcripts observed on different plasmids from the same oocyte showed differences in processing and termination efficiency, but all the RNAs on a single template behaved similarly. One explanation for this is that events at the promoter can dictate subsequent 3' processing efficiencies, which is consistent with the observations, discussed above, that CPSF can be recruited to promoters and transferred to elongating RNAP II (Dantonel et al. 1997). In any event, these studies together significantly strengthen the view that polyadenylation is functionally linked to subsequent transcription termination, but the exact mechanism remains elusive.

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