To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.

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Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.

Actin is a key regulator of RNA polymerase (pol) II transcription. In complex with specific hnRNPs, it has been proposed that actin functions to recruit pol II coactivators during the elongation of nascent transcripts. Here, we show by affinity chromatography, protein-protein interaction assays, and biochemical fractionation of nuclear extracts that the histone acetyltransferase (HAT) PCAF associates with actin and hnRNP U. PCAF and the nuclear actin-associated HAT activity detected in the DNase I-bound protein fraction could be released by disruption of the actin-hnRNP U complex. In addition, actin, hnRNP U, and PCAF were found to be associated with the Ser2/5-and Ser2-phosphorylated pol II carboxy-terminal domain construct. Chromatin and RNA immunoprecipitation assays demonstrated that actin, hnRNP U, and PCAF are present at the promoters and coding regions of constitutively expressed pol II genes and that they are associated with ribonucleoprotein complexes. Finally, disruption of the actin-hnRNP U interaction repressed bromouridine triphosphate incorporation in living cells, suggesting that actin and hnRNP U cooperate with PCAF in the regulation of pol II transcription elongation.Eukaryotic gene transcription requires dynamic alterations of chromatin structure that are mediated by chromatin remodeling complexes and histone modifying enzymes (23, 28).For transcription competence, histone acetylation allows the switch between repressive and permissive chromatin structures through direct effects on nucleosome stability and through establishment of binding sites for regulatory proteins. Considerable progress in understanding the role of histone acetylation came from the discovery that the Saccharomyces cerevisiae transcription coactivator GCN5 and, more recently, other yeast and metazoan transcription cofactors are histone acetyltransferases (HATs). Mammalian homologs of the yeast cofactor GCN5 include PCAF and GCN5L (2,27,32,33,35). PCAF and GCN5L are encoded by distinct genes, and their expressions are differential and complementary in various tissues (33,35). Moreover, GCN5L is essential for mouse development, whereas PCAF is dispensable (33, 34). Human GCN5L and PCAF form parts of distinct multiprotein HAT complexes, namely the PCAF complex (17), the TFTC complex (1), and the STAGA complex (12). While these mammalian HAT complexes are still incompletely characterized, they have related but not identical subunit compositions.PCAF/GCN5, together with the p300/CREB-binding protein, is among the best-studied transcriptional coactivators (11). PCAF has been proposed to facilitate long-distance transcriptional enhancement by direct association with enhancer sequences (7). PCAF is also known to acetylate free histones or nucleosomes, primarily on lysine 14 of histone H3 (28), and its requirement as coactivator or HAT has been demonstrated for myogenesis and nuclear receptor-mediated and growth factor-signaled activation (28). However, it is presently unclear whether PCAF is also involved in the maintenance of effic...