Wilma A. Hofmann

Nuclear actin and myosin I are required for RNA polymerase I transcription

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.

Actin is part of pre-initiation complexes and is necessary for transcription by RNA polymerase II

Actin is abundant in the nucleus and has been implicated in transcription; however, the nature of this involvement has not been established. Here we demonstrate that β-actin is critically involved in transcription because antibodies directed against β-actin, but not muscle actin, inhibited transcription in vivo and in vitro. Chromatin immunoprecipitation assays demonstrated the recruitment of actin to the promoter region of the interferon-γ-inducible MHC2TA gene as well as the interferon-α-inducible G1P3 gene. Further investigation revealed that actin and RNA polymerase II co-localize in vivo and also co-purify. We employed an in vitro system with purified nuclear components to demonstrate that antibodies to β-actin block the initiation of transcription. This assay also demonstrates that β-actin stimulates transcription by RNA polymerase II. Finally, DNA-binding experiments established the presence of β-actin in pre-initiation complexes and also showed that the depletion of actin prevented the formation of pre-initiation complexes. Together, these data suggest a fundamental role for actin in the initiation of transcription by RNA polymerase II.

SUMOylation of nuclear actin

Actin, a major component of the cytoplasm, is also abundant in the nucleus. Nuclear actin is involved in a variety of nuclear processes including transcription, chromatin remodeling, and intranuclear transport. Nevertheless, the regulation of nuclear actin by posttranslational modifications has not been investigated. We now show that nuclear actin is modified by SUMO2 and SUMO3 and that computational modeling and site-directed mutagenesis identified K68 and K284 as critical sites for SUMOylating actin. We also present a model for the actin–SUMO complex and show that SUMOylation is required for the nuclear localization of actin.

Cell and molecular biology of nuclear actin.

Actin is a highly conserved protein and one of the major components of the cytoplasm and the nucleus in eukaryotic cells. In the nucleus, actin is involved in a variety of nuclear processes that include transcription and transcription regulation, RNA processing and export, intranuclear movement, and structure maintenance. Recent advances in the field of nuclear actin have established that functions of actin in the nucleus are versatile, complex, and interconnected. It also has become increasingly evident that the cytoplasmic and nuclear pools of actin are functionally linked. However, while the biological significance of nuclear actin has become clear, we are only beginning to understand the mechanisms that lie behind the regulation of nuclear actin. This review provides an overview of our current understanding of the functions of actin in the nucleus.

Nuclear myosin I is necessary for the formation of the first phosphodiester bond during transcription initiation by RNA polymerase II

The nuclear isoform of myosin, Nuclear Myosin I (NMI) is involved in transcription by RNA polymerase I. Previous experiments showing that antibodies to NMI inhibit transcription by RNA polymerase II using HeLa cell nuclear extract (NE) suggested that NMI might be a general transcription factor for RNA polymerases. In this study we used a minimal in vitro transcription system to investigate the involvement of NMI in transcription by RNA polymerase II in detail. We demonstrate that NMI co‐purifies with RNA polymerase II and that NMI is necessary for basal transcription by RNA polymerase II because antibodies to NMI inhibit transcription while adding NMI stimulates transcription. Further investigation revealed that NMI is specifically involved in transcription initiation. Finally, by employing an abortive transcription initiation assay, we demonstrate that NMI is crucial for the formation of the first phosphodiester bond during transcription initiation. J. Cell. Biochem. 99: 1001–1009, 2006.

Nuclear actin: to polymerize or not to polymerize

The form and function of actin in the nucleus have been enigmatic for over 30 years. Recently actin has been assigned numerous functional roles in the nucleus, but its form remains a mystery. The intricate relationship between actin form and function in the cytoplasm implies that understanding the structural properties of nuclear actin is elementary to fully understanding its function. In this issue, McDonald et al. (p. 541) use fluorescence recovery after photobleaching (FRAP) to tackle the question of whether nuclear actin exists as monomers or polymers.

Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus

In vertebrates, two myosin Ic isoforms that localize to the cytoplasm and to the nucleus have been characterized. The isoform that predominantly localizes to the nucleus is called nuclear myosin I (NMI). NMI has been identified as a key factor involved in nuclear processes such as transcription by RNA polymerases I and II and intranuclear transport processes. We report here the identification of a previously uncharacterized third MYOIC gene product that is called isoform A. Similar to NMI, this isoform contains a unique N‐terminal peptide sequence, localizes to the nucleus and colocalizes with RNA polymerase II. However, unlike NMI, upon exposure to inhibitors of RNA polymerase II transcription the newly identified isoform translocates to nuclear speckles. Furthermore, in contrast to NMI, this new isoform is absent from nucleoli and does not colocalize with RNA polymerase I. Our results suggest an unexpected diversity among nuclear myosin Ic isoforms in respect to their intranuclear localization and interaction with nuclear binding partners that could provide new insights into the regulation of myosin‐dependent nuclear processes.

Ancient animal ancestry for nuclear myosin.

The identification of nuclear myosin I (NMI) has raised the possibility that myosin might have had an early functional role in the eukaryotic nucleus. To investigate this possibility, we examined the molecular evolution of the vertebrate myosin-I proteins. We found that myosin I has undergone at least five duplication events in the common ancestor of the vertebrates (vertebrate-specific duplications), leading to nine myosin-I vertebrate gene families, followed by two additional myosin-I duplication events in the lineage leading to modern fish. This expansion suggests a large-scale adaptive radiation in myosin-I function in an early phase of vertebrate evolution. The branching order of the evolutionary tree suggests that the functional role of NMI predates this expansion. More specifically, in the tunicate Ciona intestinalis, we found a myosin-I protein that localizes to the nucleus, but that branches on phylogenetic trees before the duplication that led to vertebrate myosin IC and myosin IH. This relationship suggests that the common ancestor of these three proteins encoded a nuclear isoform and that the localization of myosin I to the nucleus predates the origin of the vertebrates. Thus, a functional role for NMI appears to have been present at an early stage of animal evolution prior to the rise of both myosin IC and the vertebrates, as NMI was present in the last common ancestor of vertebrates and tunicates.

Lamin A tail modification by SUMO1 is disrupted by familial partial lipodystrophy–causing mutations

Lamin A tail domains are SUMO1 modified at K420 (nuclear localization signal) and K486 (Ig-fold). K486 modification requires Ig-fold surface residues E460 and D461 and is reduced by familial partial lipodystrophy–causing mutations G465D and K486N. These results suggest novel mechanisms of functional control over lamin A in cells.

Tissue specific expression of myosin IC isoforms.

BackgroundMyosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus and is implicated in a variety of processes in both compartments. We recently identified a novel isoform of myosin IC and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C) that differ only in the addition of short isoform-specific N-terminal peptides. The expression pattern of the isoforms and the mechanisms of expression regulation remain unknown.ResultsTo determine the expression patterns of myosin IC isoforms, we performed a comprehensive expression analysis of the two myosin IC isoforms (isoform A and B) that contain isoform-specific sequences. By immunoblotting with isoform-specific antibodies and by qRT-PCR with isoform-specific primer we demonstrate that myosin IC isoforms A and B have distinct expression patterns in mouse tissues. Specifically, we show that myosin IC isoform A is expressed in a tissue specific pattern, while myosin IC isoform B is ubiquitously expressed at comparable levels in mouse tissues.ConclusionsThe differences in the expression profile of the myosin IC isoforms indicate a tissue-specific MYOIC gene regulation and further suggest that the myosin IC isoforms, despite their high sequence homology, might have tissue-specific and isoform-specific functions.