S1-1 nuclear domains: characterization and dynamics as a function of transcriptional activity.

BACKGROUND INFORMATION: The RNA-binding protein S1-1, also called RBM10 (RNA-binding motif 10), is a paralogue of putative tumour suppressor RBM5 and has been correlated with cancer proliferation and apoptosis. In the present study, we have investigated the cell biology of S1-1. RESULTS: In the extranucleolar nucleoplasm, S1-1 occurred in hundreds of punctate and irregular domains. Some 10-40 of these domains were larger than 0.5 mum and prominent for S1-1 immunostaining. These domains (S1-1 nuclear bodies) were commonly present in tissue cells and in cultured cells. When cellular transcription was globally reduced by heat shock, serum starvation, culture at high cell densities or inhibition with RNA polymerase II inhibitors, small S1-1 domains (S1-1 granules), with weak immunostaining signals, reduced in number, whereas S1-1 nuclear bodies became prominent and increased in size. These altered S1-1 domains were returned to initial states when the cells were placed under normal conditions. Similar to paraspeckles, S1-1 nuclear bodies occurred closely adjacent to nuclear speckles or IGCs (interchromatin granule clusters), as determined by immunoelectron microscopy. However, the S1-1 nuclear bodies did not correspond to paraspeckles or IGAZs (interchromatin-granule-associated zones), but coincided with TIDRs (transcription-inactivation-dependent RNA domains), which we had characterized previously at the RNA level. The enlarged S1-1 nuclear bodies/TIDRs accumulated the S1-1 protein and microinjected primary and spliced mRNAs, presumably for later elevation of gene expression. In addition, electron microscopy revealed that S1-1 was also present on perichromatin fibrils, suggesting the structure of S1-1 granules seen at higher resolution. CONCLUSIONS: S1-1 constitutes hundreds of nuclear domains, which dynamically change their structures in a reversible manner. Upon globally reducing RNA polymerase II transcription, S1-1 nuclear bodies enlarge and decrease in number. They are novel domains different from paraspeckles or IGAZs, despite their similar occurrence adjacent to nuclear speckles. We discuss S1-1 granules in terms of their association with gene expression. In addition, this is the first report of a TIDR-localized protein.

Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells.

S1 proteins C2 and D2 are multifunctional hnRNP proteins acting as transcriptional regulators in the nucleus. Immunofluorescence staining of various cells in culture revealed that S1 proteins also occur in the cytoplasm, often in association with vimentin intermediate filaments (VFs). Here, we verified the association of S1 proteins with vimentin using vimentin-deficient cells, crosslinking and immunoprecipitation, and further investigated the biological significance of this association. S1 proteins on VFs, referred to here as S1 fibers, were lost in highly confluent cells, where cell proliferation and cellular metabolic activity greatly decreased owing to cell density-dependent arrest. However, the disappearance of S1 fibers was not related to these reduced activities, but to inhibited cell migration. Although undetected in cells of non-migratory tissues as well as in confluent cultured cells, S1 fibers were found in all migratory cells examined, such as cultured cells in scratch/wound experiments, blood neutrophils and monocytes, and fibroblasts engaging in tissue healing. In addition, S1 fibers reappeared even in confluent cells when VFs were induced to reorganize with okadaic acid. We propose that S1 proteins occur in association with VFs in migratory cells. Possible participation of S1 proteins in the formation/reorganization of VFs is discussed.

Presumptive Primordial Germ Cells (pPGCs) and PGCs in Tadpoles from UV-irradiated embryos of Xenopus: (UV-irradiation/primordial germ cell/monoclonal antibody/complete sterility/Xenopus laevis).

In order to determine whether or not tadpoles that once lacked primordial germ cells (PGCs) in the genital ridges and dorsal mesentery as a result of ultraviolet (UV) irradiation subsequently contained germ cells at more advanced stages of larval development, the numbers of presumptive PGCs or PGCs were carefully examined in Xenopus tadpoles at Nieuwkoop and Faber's stage 35/36-52 that developed normally from UV-irradiated eggs. No late-appearing germ cells were observed in almost all the UV-irradiated tadpoles examined at stages 49-52. This same population had completely lacked PGCs at about stage 46. Moreover, presumptive PGCs (pPGCs) or cells with granular cytoplasm that reacted with a monoclonal antibody specific for the germ plasm of cleaving Xenopus eggs stayed in the central part of the endoderm cell mass in the irradiated tadpoles at stage 35/36, when the majority of those cells were located in the dorsal part of the endoderm in unirradiated controls. Furthermore, in the irradiated embryos pPGCs were demonstrated to decrease in number with development and eventually to disappear in tadpoles at about stage 40. The results strongly suggest that UV irradiation under the conditions used here totally eliminated germline cells from the irradiated animals.