Search

In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)+ mRNAs in living mammalian cells using a linear antisense 2'-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1-GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2'-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)+ mRNAs. [ABSTRACT FROM AUTHOR]

Abstract: Single particle tracking (SPT) is a powerful technique for studying mRNA dynamics in cells. Although SPT of mRNA has been performed by labeling mRNA with fluorescent dyes or proteins, observation of mRNA for long durations with high temporal resolution has been difficult due to weak fluorescence and rapid photobleaching. Using quantum dots (QDs), we succeeded in observing the movement of individual mRNAs for more than 60s, with a temporal resolution of 30ms. Intronless and truncated ftz mRNA, synthesized in vitro and labeled with QDs, was microinjected into the nuclei of Cos7 cells. Almost all mRNAs were in motion, and statistical analyses revealed anomalous diffusion between barriers, with a microscopic diffusion coefficient of 0.12μm2/s and a macroscopic diffusion coefficient of 0.025μm2/s. Diffusion of mRNA was observed in interchromatin regions but not in histone2B-GFP-labeled chromatin regions. These results provide direct evidence of channeled mRNA diffusion in interchromatin regions. [Copyright &y& Elsevier]

In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu ( ftz) pre-mRNA into the nuclei of HeLa cells. The injected intron-containing ftz pre-mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy-requiring active process. In contrast, the injected intron-less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre-mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, α-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells. [ABSTRACT FROM AUTHOR]

Abstract: β-actin mRNA localizes to the leading edge of a living chicken embryo fibroblast. Recently we proposed that the mRNA maintains its localization at the leading edge by utilizing the heterogeneity of cytoplasmic microstructure (Yamagishi et al., 2009 ). In this study, we observed the intracellular distribution of β-actin mRNA variants to elucidate the mechanism of mRNA localization at the leading edge. We found that the degree of localization correlated positively with the molecular mass of the mRNA variants. We further demonstrated that the molecular mass-dependent localization was found even with dextrans, which have no biological function. The dependency of localization on molecular mass suggested that the barrier effect caused by the physical obstruction of the cytoplasmic microstructure is one of the major factors controlling mRNA localization in motile fibroblasts. [Copyright &y& Elsevier]

Abstract: β-Actin mRNA labeled with an MS2–EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most β-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D MACRO) at the leading edge was 0.3 μm2/s. On the other hand, D MACRO in the perinuclear region was 0.02 μm2/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize β-actin mRNAs, led to an increase in D MACRO to 0.2 μm2/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of β-actin mRNA. [Copyright &y& Elsevier]

The selection of effective antisense target sites on a given mRNA molecule is a major problem in the detection of target mRNA in oligonucleotide arrays. In general, antisense oligodeoxynucleotides (asODNs) of about 10–20 nucleotides (nt) in length are used. However, the demand for predicting the sequence of potent asODNs much longer than those mentioned above has been increasing. Here, we prepared 40-nt asODNs directed against fluorescence-labeled green fluorescent protein (GFP) mRNA and quantified their hybridization efficiencies by fluorescence microscopy. We found that the hybridization efficiency depended on the TC content or the minimum free energy of the asODNs. On the basis of these findings, a semiempirical parameter called accessibility score was introduced to predict the potency of asODNs. The results of this study aided in the development of an effective two-step procedure for determining mRNA accessibility, namely, the computer-aided selection of asODN binding sites using an accessibility score followed by an experimental procedure for measuring the hybridization efficiencies between the selected asODNs and the target mRNA by fluorescence microscopy. [Copyright &y& Elsevier]