On the right track

Abstract

The transcription machinery in the eukaryotic nucleus generates messenger RNA molecules that translocate through the nucleoplasm, anchor to a nuclear pore, and find their way out into the cytoplasm. The dynamic aspects of these steps in the expression pathway were examined in order to understand the kinetic time-frames of gene activation and message dissemination. Utilizing live-cell imaging and tracking of single mRNPs containing different sized mRNAs and varying numbers of introns and exons, it was possible to quantify the temporal and spatial characteristics of the nucleoplasmic travels of mRNPs as well as the kinetics of translocation through the nuclear pore.

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Acknowledgements

Y.S.T. thanks the funding agencies that supported these studies: Israel Science Foundation (grant 250/06), the Israel Science Foundation Bikura grant, United States-Israel Binational Science Foundation, Israel Cancer Research Fund, Israel Cancer Association, the Israel Ministries of Health and Science, and the Alon Fellowship. Y.S.T. is the Jane Stern Lebell Family Fellow in Life Sciences at BIU.

Figures and Tables

Figure 1 Following mRNPs in living cells. (A) Scheme of the MS2 system used for tagging mRNAs in living cells. The transcription of a gene of interest is driven by a promoter. The 3′UTR of the gene contains a series of MS2 sequence repeats, which fold into stem-loop structures in the transcribed mRNA. These are bound by YFP-MS2-CP proteins to yield the mRNP fluorescent. (B) Scheme of the travels of mRNPs from the site of transcription to the cytoplasm. Top-RNA polymerase II (green) recruited to a gene of interest generates mRNPs (red) that travel in between dense chromosomal territories (blue) and nucleoli (cyan) to the NPC. mRNPs are depicted as either unfolded or folded during transcription and translocation through the NPC. mRNPs diffuse through the nucleoplasm and can show corralled movement probably due to the hindering by the chromatin environment. Bottom-mRNA export is blocked in WGA (yellow) treated cells, and mRNPs remain anchored at the NPC.

Figure 2 Identifying nucleoplasmic mRNP tracks using maximum time projections. (A) Demonstration of the application of the maximum time projection function to time-lapse movies. Top-scheme showing the pathway of a single spot over time; bottom—single frames from a movie of YFP-MS2 labeled mRNPs. The image on the right is the maximum time projection of all the single frames. (B) Maximum time projection of mRNPs imaged for two different time periods, in the same cell shown in (A): red-maximum time projection (M t proj.) of the first 30 movie frames (captured every second); green-maximum time projection from the last 30 seconds of the movies. Hoechst DNA staining and co-localized tracks from the different times are shown. White dashed line represents one of the nuclear tracks. Right-enlargement of the boxed area. Scale bars, 5 µm.

Figure 3 Blocking mRNP export by WGA treatment. Treatment with WGA leads to an increase in peripherally stalled mRNPs. Average time projection shows the stalled mRNPs and their tracks (green, by single particle tracking), also seen in an enlarged area (yellow box, middle). An increase in membrane stalled mRNPs is seen (left - same cell before treatment) after (right) treatment with WGA. Green tracks show all static mRNPs. The rest of the mRNPs continue to move in the nucleoplasm. Schemes on right show the dynamic mRNPs (white dots) and the static mRNPs (red dots). Scale bar, 5 µm. Adapted from reference 57.

Extra View on: Mor A, Suliman S, Ben-Yishay R, Yunger S, Brody Y, Shav-Tal Y. Dynamics of single mRNP nucleo-cytoplasmic transport through the nuclear pore in living cells. Nat Cell Biol 2010; 12:543 - 552; PMID: 20453848; http://dx.doi.org/10.1038/ncb2056

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