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Dynamics and kinetics of nucleo‐cytoplasmic mRNA export

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Abstract Activation of the gene expression pathway in eukaryotic cells results in the nuclear transcription of mRNA molecules, many of which are destined for translation into protein by cytoplasmic ribosomes. mRNA transcripts are exported from the nucleus to the cytoplasm via passage through nuclear pore complexes (NPCs), ∼125 MDa supramolecular complexes set in the double‐membraned nuclear envelope. Understanding the kinetics of mRNA translocation, from the point of transcription through export, localization, translation, and degradation, is of fundamental interest since gene expression is regulated at all the different levels of this pathway. In this review, we delineate the steps taken by an mRNA molecule in transit to the nuclear envelope and during mRNA export, with specific focus on the dynamic aspects of nucleo‐cytoplasmic mRNA transport as revealed by electron microscopy and live‐cell imaging. Copyright © 2010 John Wiley & Sons, Ltd. This article is categorized under: RNA Export and Localization > Nuclear Export/Import

Schematic representation of the three‐dimensional nuclear pore complex consensus model. The main structural components of the nuclear pore complex (NPC) include the central framework, the cytoplasmic ring moiety and cytoplasmic filaments, and the nuclear ring moiety and nuclear basket. The structures of the central framework and the distal ring of the nuclear basket have been adapted from a reconstruction of native NPCs embedded in thick amorphous ice. (Reprinted with permission from Ref 1. Copyright 2003 Nature Publishing Group.)

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Calculating the time frame of mRNP nucleoplasmic translocation. (a) The cell nucleus is represented by a sphere of radius r. The nuclear pore complexes are represented by circular holes scattered at the sphere's surface. Nucleoli are represented by smaller spheres contained in the interior of the nucleus. An mRNP particle is synthesized at different distances from the nuclear pores, depending on the localization of its cognate gene template. The particle will make a 3D Pearson‐type random walk inside the nucleus, but it cannot enter nucleoli. Each random walk step has a fixed length, l. After a number of steps N, the particle will reach the nuclear pore. The relation between time and the number of steps is . (b, c) To validate that this algorithm properly described Brownian motion, the evolution of a large ensemble of particles moving without any constraints was simulated. (b) shows a histogram obtained with 1 × 104 random walking particles moving 2 × 104 steps with 0.1 (arbitrary units), showing the expected exponential decay. (c) shows that the dependence of 〈r2 〉 with number of steps N agrees with the random walk model: 〈r2 〉 = l2N. (Reprinted with permission from Ref 92. Copyright 2004 Springer.)

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Single‐particle tracking of individual mRNPs. Images from time‐lapse movies acquired from a cell transfected with the YFP‐MS2 protein for tagging of the mRNAs produced from an actively transcribing gene. (a) Tracking of mRNP particles in a transcriptionally active cell (bar, 2 µm; arrow, transcription site) showed (b) diffusing particles and (c) corralled particles. Tracks are marked in green, and time in seconds from the beginning of tracking for each particle appears in each frame. Bars, 1 µm. Analysis of tracked nucleoplasmic particles indicated the presence of three types of characterized movements: diffusive, corralled, and stationary. Directed movement was never detected. (Reprinted with permission from Ref 68. Copyright 2004 American Association for the Advancement of Science.)

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Kinetics of mRNA nucleo‐cytoplasmic transport. (a) Cells expressing YFP‐MS2 showed transcription site induction followed by the appearance of tagged mRNPs. Cells were imaged for a total of 5.5 h, every 10 min, and deconvolved frames (to enhance mRNP signal) are shown. Transcriptional induction was performed at t = 0 min. An active transcription site was seen at t = 40 min. ∼10 min later transcripts were detected in the nucleoplasm (50 min) and ∼10 min after that in the cytoplasm (60 min). (Bar, 20µm). (b) Graph representing the number of mRNPs counted in the 3D volumes of the nucleus (red) and the cytoplasm (green) throughout each frame of the movie. Plot begins at t = 40 min, the initial time point of transcription site activation. Site turns off at 260 min. (c) Three frames (t = 50, 80, 170 min) representing the increase in mRNP levels in the cell over time and the distribution of mRNPs toward the periphery of the cytoplasm. These three frames are projections of all the mRNPs in the 3D volume of the cell, presented in one frame. Therefore, mRNA signal is pronounced in the region surrounding the nucleus where the cytoplasmic volume is large. (Reprinted with permission from Ref 93. Copyright 2010 Company of Biologists.)

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Accumulation of Br‐RNA at pores in HL‐60 cells. Cells were grown in 2.5‐mM Br‐U for various times, and Br‐RNA was indirectly immunolabeled with gold particles (10 nm). Representative electron micrographs are shown on the left, and the positions of all particles in a rectangle (200 × 350 nm) over the pore were determined. The positions of all particles seen over 125 pores are indicated on the right, with a diagram (drawn at the same scale; bar, 100 nm) showing the relative positions of membrane bilayer and pore complex. In all images presented, nuclei are at the top. (a, b) After 10 min in Br‐U, only a few particles are seen. (c, d) After 30 min, more particles are concentrated over the tip of the basket, and a few are found along the inner coaxial ring or between the cytoplasmic filaments. (e, f) After 60 min, high concentrations are found over the nucleus (except the basket), and more are seen over the cytoplasm. (Reprinted with permission from Ref 52. Copyright 2000 Company of Biologists.)

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A complete cycle of Balbiani ring mRNP translocation through the nuclear pore complex (NPC) as visualized in transmission electron microscopy (TEM) (a–e) and field emission in lens scanning electron miscrcopy (FEISEM) (f–t) samples. N, C—nucleus and cytoplasm. Each vertical column of figures from left to right indicates a successive stage in transport; each horizontal row represents from top to bottom: (a–e) The export of the Balbiani ring mRNP as visualized in classical thin section studies. Initially, the mRNP binds to the distal portion of the basket (lower blue arrow in (b) and then in a sequential manner is translocated through the central NPC channel in a linear form as it is unrolled on the nuclear side (see red arrows in c–e).

The surface images (f–t) reveal this process in greater detail at the nuclear surface. (f) The nucleoplasmic entrance to the nuclear pore initially is closed by basket filaments to which the mRNP particle docks (g) and initiates the distal basket ring formation. In subsequent steps, the Balbiani ring mRNP is unrolled as it physically moves through the center of the enlarged basket ring (h, i) and finally disappears (i, j). Balbiani ring mRNP fibers have been visualized translocating through the NPC core (k–o). The inner diameter of the transporter cylinder expands from 10 nm (k) to 26 nm (l–o) during this process. The cytoplasmic transporter globule is initially closed (p, q) but forms (r), a transiently open 35‐nm ring at the base of mRNP during the translocation. A longer portion of the mRNP is visible at a later stage (s). Finally, the transporter globule is visualized (t) with a central 26‐nm pore, during either mRNP loss following isolation or mRNP exit from the central channel. mRNPs are marked by red arrows; Scale bars, 0.1 mm (b–e, same magnification; also f–t). BF, basket filaments (yellow arrows). (Reprinted with permission from Ref 49. Copyright 1998 Company of Biologists.)

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Translocation of a Balbiani ring RNP particle through the nuclear pore complex. See text for details. (Reprinted with permission from Ref 39. Copyright 1997 Elsevier.)

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Translocation of the Balbiani ring RNP particle through the nuclear pore complex. A series of electron micrographs shows the consecutive movement of the BR particle through the nuclear pore. The BR particle changes conformation during translocation, and ribosomes (arrows) can be seen associated with the RNP fiber exiting into the cytoplasm. (Reprinted with permission from Ref 48. Copyright 2001 Springer.)

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Electron micrographs of the translocation of large complexes through the NPC in (a) Xenopus laevis and (b–d)Triturus alpestris. A possible sequence of events is presented: (a) the large complex approaches the nuclear pore complex (NPC) and attaches by a thin filament; (b) it then reaches the pore center; and (c) elongates into a 100–150Å broad rod. The material passes the pore center in a rodlike form, transitorily assuming a typical dumbbell‐shaped configuration. (d) Then the material rounds into a spherical particle and is deposited on the cytoplasmic side. (A, × 83,000; B, × 135,000; C, × 110,000; D, × 100,000; bars 0.1µm). C at top of each panel = cytoplasmic side. (Reprinted with permission from Ref 35. Copyright 1974 Elsevier.)

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