Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells

Abstract

The flow of genetic information in eukaryotic cells occurs through the nucleocytoplasmic translocation of mRNAs. Knowledge of in vivo messenger RNA export kinetics remains poor in comparison with that of protein transport. We have established a mammalian system that allowed the real-time visualization and quantification of large single mRNA-protein complexes (mRNPs) during export. The in vivo dynamics of bulk mRNP transport and export, from transcription to the nuclear pore complex (NPC), occurred within a 5-40 minute time frame, with no NPC pile-up. mRNP export was rapid (about 0.5 s) and kinetically faster than nucleoplasmic diffusion. Export inhibition demonstrated that mRNA-NPC interactions were independent of ongoing export. Nucleoplasmic transport dynamics of intron-containing and intronless mRNAs were similar, yet an intron did increase export efficiency. Here we provide visualization and analysis at the single mRNP level of the various steps in nuclear gene expression and the inter-chromatin tracks through which mRNPs diffuse, and demonstrate the kinetics of mRNP-NPC interactions and translocation.

How to operate a nuclear pore complex by Kap-centric control

“…66 Nevertheless, from the perspective of Kap-centric control a displacement of preloaded Kaps from the FG Nups would result in the effective retraction/reduction of the NPC barrier. We speculate that the presence of multiple Kaps on large cargoes 67,68 (or receptors that shuttle messenger ribonucleic proteins 57,69 ) may be able to displace preloaded Kaps because of increased binding avidity with the FG Nups. Indeed, the presence of multiple Kaps has also shown to improve the transport efficiency of large cargoes.…”

SRSF1-dependent nuclear export of C9ORF72 repeat transcripts: targeting toxic gain-of-functions induced by protein sequestration as a selective therapeutic strategy for neuroprotection

“…Approximately 200 adenosines are added to the 3'-end of human mRNAs. mRNAs produced from intron-containing reporter constructs are more efficiently exported into the cytoplasm of mammalian cells [15] . In human, the vast majority of mRNA molecules are exported from the nucleus to the cytoplasm in a process that is functionally coupled to splicing via dynamic assembly of the evolutionary conserved transcription-export complex (TREX) [16] .…”

Imaging Translation in Single Cells Using Fluorescent Microscopy

“…1C) (Shav-Tal et al 2004). Transcripts diffuse within the nucleoplasm for times ranging from 5 to 40 minutes; however, once a transcript engages a nuclear pore, export into the cytoplasm is fast (less than 0.2 sec) (Grunwald and Singer 2010;Mor et al 2010). Interestingly, transport through the nuclear pore takes only 5-20 min and docking on the nuclear side and release into the cytoplasm both take approximately 80 min, indicating that translocation through the nuclear pore is not the rate-limiting step (Grunwald and Singer 2010).…”