Background:The mechanism underlying nuclear transport of RNA polymerase II (RNAPII) is unclear. Results: Npa3 is required for nuclear localization of RNAPII and binds it in a GTP-dependent manner. Conclusion: RNAPII nuclear import takes place via an unconventional pathway involving Npa3 and a cycle of GTP-dependent Npa3-RNAPII binding and release. Significance: Learning the mechanism of RNAPII nuclear import is crucial for understanding the regulation of gene expression.

The retinoblastoma family of proteins are key cell cycle regulatory molecules important for the differentiation of various mammalian cell types. The retinoblastoma protein regulates transcription of a variety of genes either by blocking the activation domain of various activators or by active repression via recruitment to appropriate promoters. We show here that the retinoblastoma family of proteins functions as direct transcriptional repressors in a heterologous yeast system when fused to the DNA binding domain of Gal4. Mapping experiments indicate that either the A or the B domain of the pocket region is sufficient for repression in vivo. As is the case in mammalian cells, a phosphorylation site mutant of the retinoblastoma protein is a stronger transcriptional repressor than the wild type protein. We show that transcriptional repression by pRb is dependent on CLN3 in vivo. Furthermore, the yeast histone deacetylase components, RPD3 and SIN3, are required for transcriptional repression.The retinoblastoma protein (pRb) 1 is a key regulatory molecule important for cell cycle control and differentiation in a number of mammalian cell types (1). p107 and p130 are two related family members, who share both sequence and functional properties with the retinoblastoma protein (2). The three proteins show greatest similarity within the so-called pocket domain, consisting of A and B subdomains. The A and B subdomains have been reported to show weak similarity to the general transcription initiation factors, TBP and TFIIB (3, 4). Evolutionarily conserved homologs of pRb have been identified in Caenorhabditis elegans (5), Drosophila (6) and plants (7), while no homologs exist in yeast.The retinoblastoma protein regulates gene expression presumably through its interaction with transcriptional activators such as E2F1 (8 -10), MyoD (11), Elf1 (12), c-Myc (13), PU.1 (4), ATF2 (14), and UBF (15). E2F1-responsive genes are repressed by pRb via the interaction of pRb with the activation domain of E2F1, and this interaction is a phosphorylation-dependent event. Phosphorylation of pRb inhibits its ability to bind the activation domain of E2F1 and the subsequent activation of E2F1-dependent transcription is sufficient for cells to proceed through cell division (16,17). Previous studies have shown that the retinoblastoma protein can be ectopically expressed in Saccharomyces cerevisiae and that cyclin-dependent phosphorylation of the protein in yeast mimics that observed in mammalian cells (18).In mammalian cells, the retinoblastoma protein bears intrinsic transcriptional repression properties when tethered to DNA via the heterologous Gal4 DNA binding domain, and this function is dependent on an intact pocket region (19 -22). The retinoblastoma protein has been shown to interact with several proteins that may be relevant to its ability to directly inhibit transcription in vivo. pRb has been shown to interact with histone deacetylase (HDAC) and presumably inhibits transcription by virtue of deacetylating chromatin in the vicinity of t...