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

Occupational Communication as Boundary Mechanism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69000/2/10.1177_073088847400100404.pd

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

Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors

The plant hormone abscisic acid (ABA) is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. Many components involved in ABA response have been identified, including the transcription factors ABA insensitive (ABI)4 and ABI5. The genes encoding these factors are expressed predominantly in developing and mature seeds, and are positive regulators of ABA mediated inhibition of seed germination and growth. The direct effects of ABI4 and ABI5 in ABA response remain largely undefined. To address this question, plants over-expressing ABI4 or ABI5 were used to allow identification of direct transcriptional targets. Ectopically expressed ABI4 and ABI5 conferred ABA-dependent induction of slightly over 100 genes in 11 day old plants. In addition to effector genes involved in seed maturation and reserve storage, several signaling proteins and transcription factors were identified as targets of ABI4 and/or ABI5. Although only 12% of the ABA- and ABI-dependent transcriptional targets were induced by both ABI factors in 11 day old plants, 40% of those normally expressed in seeds had reduced transcript levels in both abi4 and abi5 mutants. Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences. Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants

The function of actin in gene transcription

Recent developments in the field of gene transcription regulation have unfolded a key role for actin as an important co-factor for all three eukaryotic RNA polymerases. In this review article we discuss the latest findings on actin in transcription of protein-coding and ribosomal genes, in complex with specific hnRNP proteins and a form of myosin 1ß which is entirely localized to the cell nucleus. Based on these recent studies, we propose a general model where actin may function in basal gene transcription as an allosteric regulator, to recruit transcriptional co-activators on active genes. A future challenge will be the identification of the polymerization state of actin in gene transcription and how it is mechanistically regulated