RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling.

The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis. The mechanisms governing the assembly and the transport of the three eukaryotic RNA polymerases to the nucleus are in discussion. Interesting papers have demonstrated the participation of some proteins in the assembly of the nuclear RNA polymerases and in their transport to the nucleus, but the mechanisms involved are poorly understood. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols and demonstrate that the prefoldin Bud27 of Saccharomyces cerevisiae mediates the correct assembly of the three complexes prior to their translocation to the nucleus, in a process which is dependent on Rpb5. In addition, our data support the view that, during the assembly of the RNA pols, Rpb5 and Rpb6 assemble rather late compared to the rest of the complexes. Furthermore, this role of Bud27 seems to be specific, as it is not extended to other prefoldin members. Finally, the role of Bud27 seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.

The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis. Scn5a mRNA levels were determined by real-time RT–PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs. Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

Temperature-sensitive RNA polymerase III (rpc160-112and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with [³³P]orthophosphate and coextracted with [³H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25°C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37°C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: therpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25°C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

Pitx2 is a paired-related homeobox gene that has been shown to play a central role during development. In the mouse, there are three isoforms, Pitx2a, b, and c, which differ only in their amino terminal regions. Pitx2 is expressed in myotomes, myoblasts, and myofibers and may be involved in muscle patterning. However, the mechanism by which Pitx2 acts in muscle cell lineages as well as the distinct functions of the individual isoforms have not been investigated. In this study, we used Sol8 myoblasts to investigate the function of Pitx2 in skeletal myogenesis. We found that Pitx2c is the main Pitx2 isoform present in Sol8 myoblasts. Overexpression of Pitx2c in Sol8 myoblasts inhibited myocyte differentiation and myotube formation. Furthermore, Sol8 cells overexpressing Pitx2c maintained high proliferative capacity and a significant up-regulation of the cell cycle genes cyclin D1, cyclin D2, and c-myc. Gene expression analysis for Pax3 and the s MyoD and myogenin showed that Pitx2c-overexpression caused Sol8 cells to remain as myoblasts, in an undifferentiated myogenic state. Furthermore, down-regulation of the muscle-specific genes sTnI and MyHC3 demonstrated that Sol8-overexpressing Pitx2c myoblasts failed to reach terminal differentiation. This study sheds light on previously unknown functions of the Pitx2c isoform in balancing proliferation vs. differentiation in a myogenic cell line. Developmental Dynamics 235:2930–2939, 2006.