Both RNA polymerase I and II (Pol I and Pol II) in budding yeast employ a functionally homologous "torpedo-like" mechanism to promote transcriptional termination. For two well-defined Pol II-transcribed genes, CYC1 and PMA1, we demonstrate that both Rat1p exonuclease and Sen1p helicase are required for efficient termination by promoting degradation of the nascent transcript associated with Pol II, following mRNA 3 end processing. Similarly, Pol I termination relies on prior Rnt1p cleavage at the 3 end of the pre-rRNA 35S transcript. This is followed by the combined actions of Rat1p and Sen1p to degrade the Pol I-associated nascent transcript that consequently promote termination in the downstream rDNA spacer sequence. Our data suggest that the previously defined in vitro Pol I termination mechanism involving the action of the Reb1p DNA-binding factor to "road-block" Pol I transcription close to the termination region may have overlooked more complex in vivo molecular processes.[Keywords: 5Ј-3Ј exonuclease; RNA polymerase I; S. cerevisiae; transcription termination] Supplemental material is available at http://www.genesdev.org. Nuclear transcription in eukaryotes is performed by three different DNA-dependent RNA polymerases (Pol I, Pol II, and Pol III) resulting in the synthesis of ribosomal RNA (rRNA), messenger RNA (mRNA), and small noncoding RNAs (5S rRNA and tRNA). Other small RNAs (e.g., snRNAs) are synthesized by either Pol II or Pol III (Archambault and Friesen 1993). Knowledge of the molecular details that result in transcriptional initiation (e.g., through enhancer and promoter recognition) is now quite well advanced, especially for Pol II. However, the subsequent stages of the transcription cycle, elongation and termination, are less well described. In particular, the molecular process that switches each class of polymerase from processive elongation into termination is poorly understood but may differ significantly for each polymerase class. For Pol III, which copies relatively short genes, the mechanism of termination appears to be an intrinsic feature of the polymerase itself and especially the small subunit Rpc11p. Here simple sensestrand oligo(dT) sequences appear to define Pol III termination sites (Braglia et al. 2005). In marked contrast, the mechanism of Pol II termination is far more complex, being intimately connected to processing of the premRNA. Thus capping, splicing, and cleavage/polyadenylation of the pre-mRNA all occur cotranscriptionally, mediated by mRNA processing factors that are known to interact directly or indirectly with the Pol II large subunit, the C-terminal domain (CTD) region, depending on its phosphorylation state (Orphanides and Reinberg 2002). Significantly, Pol II termination is dependent on pre-mRNA 3Ј end processing and requires a specific set of termination factors and complex genetic signals (Proudfoot 2004;Buratowski 2005). Additionally, Pol II termination requires at least some of the cleavage/polyadenylation factors that, as well as processing the premRNA, may el...

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

SummaryTranscription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3′ end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.

The c-myc proto-oncogene encodes a transcription factor that promotes cell cycle progression and cell proliferation, and its deficiency results in severely retarded proliferation rates. The ATF3 stress response gene encodes a transcription factor that plays a role in determining cell fate under stress conditions. Its biological significance in the control of cell proliferation and its crosstalk regulation, however, are not well understood. Here, we report that the serum response of the ATF3 gene expression depends on c-myc gene and that the c-Myc complex at ATF/CREB site of the gene promoter plays a role in mediating the serum response. Intriguingly, ectopic expression of ATF3 promotes proliferation of c-myc-deficient cells, mostly by alleviating the impeded G1-phase progression observed in these cells, whereas ATF3 knockdown significantly suppresses proliferation of wild-type cells. Our study demonstrates that ATF3 is downstream of the c-Myc signaling pathway and plays a role in mediating the cell proliferation function of c-Myc. Our results provide a novel insight into the functional link of the stress response gene ATF3 and the protooncogene c-myc.

No abstract