Splicing-dependent RNA polymerase pausing in yeast.

In eukaryotic cells, there is evidence for functional coupling between transcription and processing of pre-mRNAs. To better understand this coupling, we performed a high-resolution kinetic analysis of transcription and splicing in budding yeast. This revealed that shortly after induction of transcription, RNA polymerase accumulates transiently around the 3' end of the intron on two reporter genes. This apparent transcriptional pause coincides with splicing factor recruitment and with the first detection of spliced mRNA and is repeated periodically thereafter. Pausing requires productive splicing, as it is lost upon mutation of the intron and restored by suppressing the splicing defect. The carboxy-terminal domain of the paused polymerase large subunit is hyperphosphorylated on serine 5, and phosphorylation of serine 2 is first detected here. Phosphorylated polymerase also accumulates around the 3' splice sites of constitutively expressed, endogenous yeast genes. We propose that transcriptional pausing is imposed by a checkpoint associated with cotranscriptional splicing.

p73 is a p53-independent, Sp1-dependent repressor of cyclin B1 transcription.

The p53 protein family, comprised of p53, p63, and p73, has an important role in controlling cell growth and differentiation. We have previously reported that p53 prevents G(2)/M transition by decreasing intracellular levels of both cyclin B1 mRNA and protein, and attenuating the activity of the cyclin B1 promoter. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that a cyclin B1-dependent G(2) checkpoint has a role in preventing neoplastic transformation. There is high sequence similarity between p73 and p53, suggesting that the two may have similar ability to repress transcription. In this report, we find that expression of p73alpha and p73beta isoforms can decrease the levels of cyclin B1 mRNA and attenuate expression from the cyclin B1 promoter. This attenuation occurs in both p53-deficient and p53-containing cell lines and cannot be inhibited by a p53 variant deficient in repressing cyclin B1 promoter activity. p73-mediated attenuation of the cyclin B1 promoter is dependent on the presence of functional Sp1-binding sites and is independent of the NF-Y-binding sites. This suggests that p73 mediates transcriptional repression through the Sp1 transcription factor.

p53 is a NF-Y- and p21-independent, Sp1-dependent repressor of cyclin B1 transcription.

The p53 tumour suppressor protein is a DNA-binding transcription factor activated in response to DNA damage. Inactivation of the p53 gene occurs in 40-60% of human tumours and a substantial body of work indicates that loss of p53 activity is a critical step in oncogenesis. p53 helps to protect against neoplasia by inducing death in cells that have sustained irreparable DNA damage or by blocking cell cycle progression to allow time for DNA repair. We have previously reported that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter [S.A. Innocente, J.L. Abrahamson, J.P. Cogswell, J.M. Lee, p53 regulates a G2 checkpoint through cyclin B1, Proc. Natl. Acad. Sci. USA 96 (1999) 2147-2152]. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that a cyclin B1-dependent G2 checkpoint has a role in preventing neoplastic transformation. Here, we show that p53-mediated attenuation of the cyclin B1 promoter occurs in both p21+/+ and p21-/- cell lines. Furthermore, promoter attenuation is dependent on the presence of functional Sp1 binding sites and is independent of the NF-Y binding sites. We also find that wild-type, but not mutant, p53 protein binds Sp1 and the cyclin B1 promoter. This suggests that wild-type p53 mediates transcriptional repression of cyclin B1 through the Sp1 transcription factor.