Dynamic changes of nuclear RNA foci in proliferating DM1 cells

Abstract

Nuclear RNA foci are molecular hallmarks of myotonic dystrophy type 1 (DM1). However, no designated study has investigated their formation and changes in proliferating cells. Proliferating cells, as stem cells, consist of an important cellular pool in the human body. The revelation of foci changes in these cells might shed light on the effects of the mutation on these specific cells and tissues. In this study, we used human DM1 iPS-cell-derived neural stem cells (NSCs) as cellular models to investigate the formation and dynamic changes of RNA foci in proliferating cells. Human DM1 NSCs derived from human DM1 iPS cells were cultured under proliferation conditions and nonproliferation conditions following mitomycin C treatment. The dynamic changes of foci during the cell cycle were investigated by fluorescence in situ hybridization. We found RNA foci formed and dissociated during the cell cycle. Nuclear RNA foci were most prominent in number and size just prior to entering mitosis (early prophase). During mitosis, most foci disappeared. After entering interphase, RNA foci accumulated again in the nuclei. After stopping cell dividing by treatment of mitomycin C, the number of nuclear RNA foci increased significantly. In summary, DM1 NSC nuclear RNA foci undergo dynamic changes during cell cycle, and mitosis is a mechanism to decrease foci load in the nuclei, which may explain why dividing cells are less affected by the mutation. The dynamic changes need to be considered when using foci as a marker to monitor the effects of therapeutic drugs.

Fig. 1

Nuclear RNA foci number varies among DM1-03 NSCs cultured in proliferating medium. Occasionally, cytoplasmic foci are seen. A. DAPI only. B. merged picture with microtubules (Alpha-tubulin) staining of cytoskeleton. Most of the cells in this field are in interphase (lacking architecture of spindle fibers of mitosis)

Fig. 2

Nuclear RNA foci undergo dynamic changes during cell cycles. In this set of figures, cytoskeleton is stained green with alpha-tubulin to show the structure of mitosis. In early prophase (A), cells start rounding up. Nucleolus fades. Chromatin condenses to form chromosomes. Cells contain more nuclear RNA foci and no foci are seen in cytoplasm. In late prophase (or prometaphase) (B), spindle fibers form. Nuclear envelop starts breaking down. Foci are seen released from nucleus (appearing outside of DAPI area). In metaphase (C, D), chromosomes align with their centromeres on the equator across the center of the cell, spindle fibers attach to centromere. The nuclear envelop disappears. Foci vary in size and number but in general are dissipating. Through anaphase (E) and telophase (F), chromatids of each chromosome separate into daughter chromosomes and move to opposite poles. Remaining foci are randomly distributed between the two daughter cells. In cytokinesis (G, H), cell membrane pinches in around the middle of the cells. A contractile ring cleaves the cells into two daughter cells. Foci drop in number and size and remaining foci are randomly distributed to daughter cells at the end of this stage. After cytokinesis, microtubules reorganize into a new cytoskeleton for the return to interphase. A new nuclear envelop forms around each region of chromosomes. Foci can be trapped in the nucleus or left in the cytoplasm (I). If cells get out of cell cycle, foci will further accumulate and grow larger (J)

Fig. 3

Scatter plot to show nuclear RNA foci number reaches the highest just before entering mitosis (prophase). During mitosis (metaphase, anaphase and telophase), nuclear RNA foci number drops. If cells get out of cell cycle (G0), nuclear RNA foci further accumulate

Fig. 4

Dying cell (squared) is shown with dissembled nuclei and abundant nuclear RNA foci (A, B) in comparison to dividing DM1 NSCs identified by nestin staining (C). No foci are seen in normal iPS-cell derived NSCs (D).

Fig. 5

Nuclear RNA foci start accumulating in number and size with time after they stopped dividing. A, B. DM1 NSCs cultured in proliferating condition. C-G. 8 days after Mitomycin C treatment. More and larger foci are seen in the nuclei. The biggest foci reach to 2μm in diameter (F). In some cells with fewer foci, large foci tend to localize to one pole of the cells (E, G). No cytoplasmic foci were seen on day 8 after Mitomycin C treatment. B, D: pictures were taken with some green background to show the cell contour

Fig. 6

Nuclear RNA foci had significantly increased in number (A) and area (B) on day 8 after being stopped dividing by treatment with Mitomycin C (P<0.01)