Rie Mizuno

MALAT‐1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility‐related genes

MALAT‐1, a long non‐coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short‐interfering RNA‐mediated MALAT‐1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT‐1 knockdown cells, pre‐mRNA levels were decreased in some but not all genes. Thus, our findings suggest that MALAT‐1 is a novel class of non‐coding RNA that promotes cell motility through transcriptional and post‐transcriptional regulation of motility related gene expression.

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles

MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.

Formation of tRNA granules in the nucleus of heat-induced human cells

The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells

Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNAMet (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

Use of an otolith-deficient mutant in studies of fish behavior in microgravity.

The mutant strain (ha) of medaka (Oryzias latipes) lack utricular otoliths as fry, and some never form otoliths for life. The cross (F1 generation) between the strain having good eyesight and another strain having ordinary eyesight augmented visual acuity of the F1 generation. Crossing the good eyesight strain and ha mutant produced fish having good eyesight and less sensitivity to gravity in the F2 population. Their tolerance to microgravity was tested by parabolic flight using an airplane. The fish exhibited less looping and no differences in degree of looping between light and dark conditions, suggesting that loss of eyesight (in darkness) is not a direct cause for looping behavior in microgravity. The ha embryos could not form utricular otoliths. They did form saccular otoliths, but with a delay. Fry of the mutant fish lacking the utricular otoliths are highly dependent on light upon hatching and exhibit a perfect dorsal-light response (DLR). As they grow, they eventually shift from being light-dependent to being gravity-dependent. Continuous treatment of the fry with altered light direction suppressed this shift to gravity dependence. Being less dependent on gravity, these fish can serve as models in studying the differences expected for the vestibular system of fish reared in microgravity. When these fish were exposed to microgravity (parabolic flights) of an airplane, they spent far less time looping than fish reared in an ordinary light regimen.

Otolith formation in a mutant Medaka with a deficiency in gravity sensing

Mutant Medaka ha exhibit spontaneous mutation that is characterized by frequent inhibition or perturbation in the formation of utricular otoliths and/or semicircular canals. Three major features of otolith morphogenesis were observed in ha strain: 1) The initial appearance of otoliths was delayed, mispositioned, and malformed compared to normal embryos. 2) No utricular otoliths appeared on macula of any ha fry just after hatching. A symmetric state of otoliths was seen only when saccular otoliths were situated on macula in both inner ears. 3) In some fry, formation of utricular otoliths was observed in their later development. However, no new utricular otoliths appeared after fish were seventy or more days old after hatching. These observations show that otolith morphogenesis in ha is very different from that of wild-type. In this study, we classified adult ha into four different phenotypes using the existence or absence of utricular otoliths as our criteria. We concluded that dysfunction of utricular otoliths and semicircular canals cause a defect that affects the gravity-sensing abilities of medaka ha.