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Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

As previous studies suggested, nuclear myosin I (NMI) and actin have important roles in DNA transcription. In this study, we characterized the dynamics of these two proteins during transcriptional activation in phytohemagglutinin (PHA) stimulated human lymphocytes. The stimulation led to strong up-regulation of NMI both on the mRNA and protein level, while actin was relatively stably expressed. The intranuclear distribution of actin and NMI was evaluated using immunogold labeling. In nucleoli of resting cells, actin was localized predominantly to fibrillar centers (FCs), while NMI was located mainly to the dense fibrillar component (DFC). Upon stimulation, FCs remained the main site of actin localization, however, an accumulation of both actin and NMI in the DFC and in the granular component was observed. In the nucleoplasm of resting lymphocytes, both actin and NMI were localized mostly in condensed chromatin. Following stimulation, the majority of both proteins shifted towards the decondensed chromatin. In transcriptionally active cells, both actin and NMI colocalized with nucleoplasmic transcription sites. These results demonstrate that actin and NMI are compartmentalized in the nuclei where they can dynamically translocate depending on transcriptional activity of the cells.

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.