Temporal and spatial regulation of mRNA export: Single particle RNA-imaging provides new tools and insights

Abstract

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression.

Keywords: MS2-MCP system; PP7-PCP system; RNA-binding protein; in vivo single molecule imaging; mRNA export; nuclear pore complex.

Figure 1

Single molecule techniques can overcome limitations in understanding cellular mRNP function obtained from ensemble composition data from large-scale sequencing and proteomic approaches. A: Single molecule fluorescence in situ hybridization (smFISH) can reveal localization, abundance and cell-to-cell variability of individual mRNPs, but is a static method that does not allow visualization of in vivo dynamics, such as nuclear export kinetics or transport directionality of single transcripts. B: Single molecule imaging (SMI) enables a dynamic profiling of individual transcripts in vivo and provides spatial information on RBP function with high temporal resolution. C: Large-scale approaches analyzing RBP binding to mRNA transcripts are based on numerous data points that are compiled into an ensemble average, which may not be representative of individual mRNP composition in vivo. For example, the binding profile of an RBP may differ substantially across transcripts from different genes and/or transcripts residing in different cellular compartments.

Figure 2

Regulating gene expression: A role for the nuclear periphery and NPCs? A: Transcriptional activation (blue with yellow arrow) and silencing (green with black arrow) have both been associated with peripheral gene positioning. The molecular basis for such chromatin rearrangements and the local environment this places a gene in to alter gene expression remains largely unknown, as does the impact gene tethering has on mRNA export. B: SMI techniques using multi-camera setups can determine the dynamic position of a transcript relative to its gene locus in different cellular conditions. C: Genome organization may also affect the transport routes taken by different classes of RNAs. rRNA export might be biased to NPCs associated with the nucleolus (blue), and mRNA export even disfavored, due to the different biophysical properties of the nucleolus or function of nearby NPCs (exemplified by the lack of Mlp1/2 in NPCs close to the nucleolus). Other genomic regions might play a similar role by facilitating local environments that select for distinct mRNPs. D: SMI can unravel such gene expression features through differential labeling of sub-nuclear structures and/or different classes of mRNAs.

Figure 3

Models of Dbp5-dependent directional mRNA export. A: Schematic of a ‘scaffold’ model of Dbp5 dependent export. Dbp5 is recruited to the mRNA in the nucleus and incorporated into the mRNP to generate a platform for other proteins to bind. Dbp5 travels through the pore with the mRNP and upon reaching the cytoplasmic side of the NPC remodels the mRNP though its ATPase activity due to the presence of regulators (e.g. Gle1:InsP6 and Nup159). Dbp5 is then recycled back to the nucleus. B: Using SMI techniques, individual mRNPs would be expected to show nuclear maturation and/or docking defects in the ‘scaffold’ scenario. C: Schematic of a ‘Brownian ratchet’ model of Dbp5 dependent export. Dbp5 waits at the cytoplasmic side of the NPC for a translocating mRNP with regulators (e.g. Gle1:InsP6 and Nup159) and continuously remodels the mRNP through its ATPase activity. D: In contrast to the ‘scaffold’ model, individual mRNPs in the ‘ratchet’ scenario would be expected to show cytoplasmic release defects and/or mRNP re-import using SMI.