New insights into the mechanism of mRNA export in living cells using quantitative microscopy approaches

Eukaryotic cells have developed a fascinating mechanism to ensure that the genetic information is transmitted from the nuclear side of transcription to the cytoplasmic side of protein production. This process is called messenger RNA (mRNA) export and is responsible for the expression of all nuclear encoded genes in eukaryotic cells. Mutations affecting the nuclear transport machinery can lead to developmental and neurological disorders or have been implicated in carcinogenesis. Furthermore, also viruses highjack the mRNA export machinery for their own survival and replication. Although the process of mRNA export is essential and highly conserved, the detailed underlying mechanism remains poorly understood. Up to now, most of the performed studies have focussed on biochemical or genetic experiments, which lack the dynamic context of the living cell. Due to the limited in vivo information available to date, the temporal and spatial regulation of mRNA export and the interactions of export factors with the mRNA still remain to be unravelled. To study the complex mechanism of real-time mRNA export in vivo, an organism allowing straightforward genetic manipulations and microscopy approaches is needed. The yeast species Saccharomyces cerevisiae is particularly suitable since it offers a fast generation time and the possibility to easily modify endogenous genes. In this thesis, I developed novel approaches to investigate the mechanism of mRNA export in living S. cerevisiae cells under physiological conditions. Using fluorescence cross-correlation spectroscopy, I examined which central export factors interact with the mRNA already in the nucleus. In addition, data from single molecule imaging suggests that mRNA export times in yeast may be similar to those in mammalian cells. Furthermore, to understand the underlying mechanism of mRNA export and translocation at the nuclear pore complex (NPC) in detail, the currently proposed ‘scaffold model’ and the ‘ratchet model’ were tested. The ‘scaffold model’ suggests that Dbp5, an essential mRNA export and remodelling factor, is exported together with the mRNA from the nucleus into the cytoplasm. The ‘ratchet model’, on the other hand, proposes that Dbp5 remains at the cytoplasmic side throughout the entire export process, thereby being responsible for the unloading of export factors such as Mex67. To differentiate between these two hypotheses, I investigated the behaviour of Dbp5, Mex67 and their binding partners, including their localization and dynamics at the nuclear pore using quantitative imaging experiments in combination with fluorescence recovery after photo-bleaching. The accumulated data were not fully consistent and compatible with either of the known models, therefore I propose a novel model for mRNA export termed the ‘stripping model’. Taken together, the work presented in this thesis gives new insight into the process of mRNA export, covering early steps of nuclear mRNA processing, the export through the NPC itself, as well as the mRNA remodelling events at the cytoplasmic side of the NPC. In addition, detailed in vivo data hints towards a new mRNA export model, revealing the important role of the nuclear pore complex during mRNA export

Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

ISSN:0027-8424ISSN:1091-649

Glucose stress causes mRNA retention in nuclear Nab2 condensates

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This co-incides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.ISSN:2666-3864ISSN:2211-124

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p.

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼ 200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport

The RNA export factor Mex67 functions as a mobile nucleoporin

International audienceThe RNA export factor Mex67 is essential for the transport of mRNA through the nuclear pore complex (NPC) in yeast, but the molecular mechanism of this export process remains poorly understood. Here, we use quantitative fluorescence microscopy techniques in live budding yeast cells to investigate how Mex67 facilitates mRNA export. We show that Mex67 exhibits little interaction with mRNA in the nucleus and localizes to the NPC independently of mRNA, occupying a set of binding sites offered by FG repeats in the NPC. The ATPase Dbp5, which is thought to remove Mex67 from transcripts, does not affect the interaction of Mex67 with the NPC. Strikingly, we find that the essential function of Mex67 is spatially restricted to the NPC since a fusion of Mex67 to the nucleoporin Nup116 rescues a deletion of MEX67 Thus, Mex67 functions as a mobile NPC component, which receives mRNA export substrates in the central channel of the NPC to facilitate their translocation to the cytoplasm