Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

Glycogen Synthase Kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation

Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus

The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases

Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must efficiently remove their substrates throughout the cells in a timely manner as many of these proteins can be toxic. This study shows that proteasomes can do so efficiently because they are highly mobile. Furthermore this study uncovers that proteasome mobility requires functional Arc3, a subunit of the Arp2/3 complex

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1α), and other RDM1 isoforms. We show that RDM1α undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response

Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I

Background: Human RNase P has been initially described as a tRNA processing enzyme, consisting of H1 RNA and at least ten distinct protein subunits. Recent findings, however, indicate that this catalytic ribonucleoprotein is also required for transcription of small noncoding RNA genes by RNA polymerase III (Pol III). Notably, subunits of human RNase P are localized in the nucleolus, thus raising the possibility that this ribonucleoprotein complex is implicated in transcription of rRNA genes by Pol I. Methodology/Principal Findings: By using biochemical and reverse genetic means we show here that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein subunits for destruction by RNA interference or its H1 RNA moiety for specific cleavage causes marked reduction in transcription of rDNA by Pol I. However, RNase P restores Pol I transcription in a defined reconstitution system. Nuclear run on assays reveal that inactivation of RNase P reduces the level of nascent transcription by Pol I, and more considerably that of Pol III. Moreover, RNase P copurifies and associates with components of Pol I and its transcription factors and binds to chromatin of the promoter and coding region of rDNA. Strikingly, RNase P detaches from transcriptionally inactive rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process that is correlated with renewal of transcription

Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I

Human RNase P has been initially described as a tRNA processing enzyme, consisting of H1 RNA and at least ten distinct protein subunits. Recent findings, however, indicate that this catalytic ribonucleoprotein is also required for transcription of small noncoding RNA genes by RNA polymerase III (Pol III). Notably, subunits of human RNase P are localized in the nucleolus, thus raising the possibility that this ribonucleoprotein complex is implicated in transcription of rRNA genes by Pol I.By using biochemical and reverse genetic means we show here that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein subunits for destruction by RNA interference or its H1 RNA moiety for specific cleavage causes marked reduction in transcription of rDNA by Pol I. However, RNase P restores Pol I transcription in a defined reconstitution system. Nuclear run on assays reveal that inactivation of RNase P reduces the level of nascent transcription by Pol I, and more considerably that of Pol III. Moreover, RNase P copurifies and associates with components of Pol I and its transcription factors and binds to chromatin of the promoter and coding region of rDNA. Strikingly, RNase P detaches from transcriptionally inactive rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process that is correlated with renewal of transcription.Our findings reveal that RNase P activates transcription of rDNA by Pol I through a novel assembly process and that this catalytic ribonucleoprotein determines the transcription output of Pol I and Pol III, two functionally coordinated transcription machineries

Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review

Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations