To the pore and through the pore: a story of mRNA export kinetics

Abstract

The evolutionary 'decision' to store genetic information away from the place of protein synthesis, in a separate compartment, has forced eukaryotic cells to establish a system to transport mRNAs from the nucleus to the cytoplasm for translation. To ensure export to be fast and efficient, cells have evolved a complex molecular interplay that is tightly regulated. Over the last few decades, many of the individual players in this process have been described, starting with the composition of the nuclear pore complex to proteins that modulate co-transcriptional events required to prepare an mRNP for export to the cytoplasm. How the interplay between all the factors and processes results in the efficient and selective export of mRNAs from the nucleus and how the export process itself is executed within cells, however, is still not fully understood. Recent advances in using proteomic and single molecule microscopy approaches have provided important insights into the process and its kinetics. This review summarizes these recent advances and how they led to the current view on how cells orchestrate the export of mRNAs. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

Figure 1. Co-transcriptional assembly of an export competent mRNP

The assembly of an export competent mRNP is orchestrated by the coordinated a recruitment of mRNA export factors to the nascent mRNA. The THO complex is recruited to the polymerase complex by an unknown interaction with the polymerase and ensures that the nascent mRNA is not forming DNA-RNA hybrids. It also recruits Sub2. The 3’ processing factor Pcf11 binds to the Ser2 phosphorylated CTD and brings Yra1 to the 3’ end of the gene where it is transferred to Sub2. Npl3 is also deposited on the mRNA during 3’ end formation. Only fully processed mRNPs can leave the site of transcription, and export competency is monitored by the nuclear exosome component Rrp6.

Figure 2. Timescales and distances for different steps of the mRNA export process in yeast and vertebrates

A. Cartoon showing the gene expression pathway. Transcription, nuclear diffusion and cytoplasmic lifetimes differ significantly between lower and higher eukaryotes, mRNP translocation through the pore, however, is likely to be similar in all organisms. Timescales in yeast are shown in red, timescales in vertebrates in green. B. Comparison of nuclear size in yeast and in vertebrates. Diffusion of an mRNP to the nuclear periphery by Brownian motion will require significantly more time in cells with a larger nucleus.

Figure 3. Recruitment of mRNPs to the nuclear pore

A. Schematic representation of the nuclear pore complex. The nuclear pore complex is a cylindrical structure comprised of eight spokes surrounding a central tube, a basket extending into the nucleus and cytoplasmic fibrils. The central transport tube (or channel) is filled with FG nups that interact with transport receptors and mediate the translocation trough the channel (see text for details). Dimensions of yeast (green) and vertebrate (red) NPC are shown. Illustrations reprint with permission from S. Patel (http://sspatel.googlepages.com/nuclearporecomplex) B. Cartoon showing NPC interactions relevant for mRNA export. Different genes in yeast were shown to be tethered to the NPC by the TREX-2 complex. Interaction of mRNPs with the NPC are mediated by the interaction of the basket protein Mlp1 with the RNA-binding protein Nab2. Before entering the pore, modification of Yra1 by the ubiquitin modifying enzyme Tom1 leads to the dissociation of Yra1 from the mRNP, one possible trigger to allow the mRNP access to the nuclear pore. An alternative access might be mediated by the Nup84 complex, forming the outer ring of the central structure of the NPC and directly interacting with the export receptor Mex67 (see text for details).

Figure 4. Real-time observation of scanning, docking and translocation of an individual mRNP

A. Description of the MS2 system, the most commonly used method for single molecule mRNA detection for live cell imaging. The system uses a bacteriophage RNA binding protein fused to GFP that binds a specific RNA stem loop structure with high affinity. Adding multiple binding sites for to an mRNA sequence allows single mRNA detection. B. Observation of mRNA export in real time. Images show single beta-actin mRNAs at different stages of mRNA export (from Grünwald and Singer, 2010 [1]). Single mRNAs are observed scanning the nuclear periphery, static at the NPC or translocating to the cytoplasm. Images were acquired at frame rates of 20ms per frame. Frames are shown in the upper left corner of each image. ‘Max’ shows the sum of all previous frames. Nuclear pore complexes are labeled in red, beta-actin mRNAs in green. N=nucleus, c=cytoplasm. Reprint with permission of Nature Publishing Group.

Figure 5. mRNP release

The ATP dependent RNA helicase/ATPase Dbp5 plays a crucial role in the removal of Nab2 and Mex67-Mtr2 from the poly(A)mRNAs and the release of mRNAs into the cytoplasm. When the mRNP reaches the cytoplasmic site of the nuclear pore, the formation of a Gle1-IP6-Dbp5-RNA complex induces the ATPase activity of Dbp5. This leads to a conformational change in Dbp5 and ATP hydrolysis, inducing the dissociation of Mex67 and Nab2 from the mRNP and subsequent release of the mRNA into the cytoplasm (see text for details). Nu=nucleus, CP= cytoplasm.