Dynamics of single mRNP nucleocytoplasmic transport and ... - Nature

A RT I C L E S

Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells Amir Mor1, Shimrit Suliman1, Rakefet Ben-Yishay1, Sharon Yunger1, Yehuda Brody1 and Yaron Shav-Tal1,2 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. The NPC machinery and auxiliary factors involved in mRNA export are well established biochemically 1–3, whereas the in vivo kinetics of mRNA export at the single-molecule level remain uncharacterized4,5. Studies with high-resolution electron microscopy on fixed Chironomus tentans specimens identified uniquely large mRNPs — the Balbiani ring granules (about 30 kilobases (kb)) — in transit through the NPC6. This approach revealed mRNP binding to fibres on the nuclear side, followed by unfolding and translocation 5´-end-first through the channel7. Sequential temporal information is lacking in such studies, because fixed specimens were used8–12. Radiolabelling experiments in purified nuclei, erythroid cells, microinjected in vitro-transcribed mRNAs and viral reporters have been used to measure mRNA nucleo-cytoplasmic transport and bulk export rates. Measured nucleocytoplasmic mRNA translocation times have ranged from minutes to several hours13–23, although slow kinetics of 1 h or more probably do not represent the situation in vivo22. In many of these experimental systems, mRNA export was uncoupled from transcription and splicing. Certain nuclear export signals are conveyed by factors bound co-transcriptionally to the mRNA; the use of exogenous unprocessed mRNAs therefore cannot fully simulate naturally assembled mRNPs. As a result of a lack of appropriate live-cell systems it has not been possible to quantify the in vivo translocation kinetics of a specific individual mRNP during export. In this study, using a transcription-inducible system, large mRNPs were followed in single living cells throughout nucleoplasmic travels and NPC translocation. We quantified the kinetic properties of single mRNPs before and during export, namely the kinetics of mRNA–NPC interactions, the possibility of mRNA pile-up at the 1 2

pore, mRNP restructuring and unfolding, the time frame of mRNA export, and the interchromatin tracks through which mRNPs move. Our results shed light on the connection between mRNA splicing, mRNA size and nuclear architecture during mRNA translocation and export. Results A cell system for monitoring mRNP nucleocytoplasmic transport and export To characterize in vivo export kinetics at the single mRNP level, and to understand the relative temporal contribution of export to the total kinetics of nucleocytoplasmic translocation, we established a mammalian cell system harbouring stably integrated genes that enabled the tracking of single mRNPs from the time of transcription and during nuclear transport and export. Previously, mRNPs have been tracked in the nucleoplasm with a series of MS2 stem-loops inserted in the 3´ untranslated region (UTR) of the gene of interest, in conjunction with the binding of yellow fluorescent protein (YFP)-tagged MS2 proteins to these structures, to yield single mRNP labelling in vivo24. Although these mRNPs (2.4 kb; ref. 25) reached the cytoplasm, single mRNP export events were not detected by live-cell imaging, and comparison with other mRNA species was difficult because of differences between cell systems. We therefore generated a series of comparable genes that transcribe large mRNAs. We speculated that these would have slower transport kinetics and might require a restructuring period during which the mRNP would unfold to facilitate NPC translocation, as observed by electron microscopy for large mRNPs6,8.

The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan 52900, Israel. Correspondence should be addressed to Y.S.-T. (e-mail: [email protected])

Received 11 March 2010; accepted 15 April 2010; published online 9 May 2010; DOI:10.1038/ncb2056

nature cell biology VOLUME 12 | NUMBER 6 | JUNE 2010 © 2010 Macmillan Publishers Limited. All rights reserved.

543

A RT I C L E S a

YFP–MS2

c Cer probe (Cy5)

MS2 probe (Cy3)

Merge + DNA

Intron probe

Merge + DNA

mRNA HSP Minimal Promoter

HSP Minimal Promoter

Cerulean-Mini-Dys

Cerulean½ Mini-Dys

Cerulean½ Mini-Dys + intron

MS2 repeats ×24

3′ UTR

Full Dys

MS2 repeats ×24

Intron



MS2 repeats ×24

GFP–Dys

MS2 repeats ×24

Mini-Dys

b ch-MS2

Full Dys

Anti- Pol II

DNA

½-Mini-Dys

Mini-Dys ½-Mini-Dys + intron

d ½-Mini-Dys

½-Mini-Dys + intron

Cer probe


Figure 1 A mammalian cell system expressing large mRNAs. (a) Scheme of the four gene constructs containing an inducible promoter and a gene encoding an mRNA with the following modules: fluorescent protein fused to dystrophin-coding regions (pink), followed by a 3´ UTR containing 24 MS2 sequence repeats (yellow). The fourth construct contains an intron (purple). The transcribed mRNA is coated with YFP–MS2 proteins (yellow balls) for detection in live-imaging experiments. All constructs are under inducible control (grey; mHSP, minimal heat shock promoter). (b) Endogenous Pol II (green) detected

by immunofluorescence, is recruited to the active transcription sites detected with mCherry-MS2 protein (ch-MS2, red). Hoechst DNA stain is in blue. (c) RNA-FISH with probes to the 5´-Cerulean (green) and 3´-MS2 (red) regions of the transcribed mRNAs, detected the mRNAs from all 4 genes after PonA induction. Transcription site signals have been intensified so that the signal of the cellular mRNAs can be seen. (d) RNA-FISH to the Cerulean exonic region (green) and the intron sequence (red) of the ½-Mini-Dys+intron mRNA. PremRNA is detected at the transcription site only. Scale bars, 10 µm.

Gene constructs containing different forms of the human dystrophin (Dys) cDNA fused to a fluorescent protein (green fluorescent protein (GFP) or Cerulean (Cer)) were generated: Full-Dys26 (14 kb mRNA including tag sequences), Mini-Dys27 (8 kb mRNA) and a smaller version, ½-Mini-Dys (4.8 kb) with or without an intron (Fig. 1a). The genes were cloned under inducible promoter control based on the Drosophila ecdysone receptor (Supplementary Information, Fig. S1a). Human U2OS cells stably expressing the ecdysone receptor were stably transfected with either of the Dys genes. Gene expression was induced by ponasterone A (PonA) and positive cell clones showing cytoplasmic Dys expression were isolated (Supplementary Information, Fig. S1b). Immunofluorescence showed that endogenous RNA polymerase II (Pol II) was recruited to the active transcription sites (Fig. 1b). RNA-fluorescence in situ hybridization (FISH) with probes to the 5´ end (fluorescent-protein region) or the 3´ end (MS2 region) of the mRNA demonstrated that the large mRNAs were fully expressed, because both transcription sites

and cellular mRNAs were detectable (Fig. 1c). The intron sequence was detected only at the transcription site of the ½-Mini-Dys+intron gene (Fig. 1d), implying that co-transcriptional splicing was occurring before the release of the mature mRNA. The ½-Mini-Dys+intron premRNA was correctly spliced, and mRNA accumulation was quantified (Supplementary Information, Fig. S2). Calibration of PonA activation over time by RNA-FISH showed that times from 4 h after treatment with PonA were suitable for following nucleoplasmic mRNAs in single cells (Supplementary Information, Figs S3 and S4). Active transcription sites and single mRNPs were detected by transient transfection with YFP–MS2-NLS (nuclear localization sequence). To improve the detection of transcriptional induction and mRNP translocation, we generated two versions of the YFP–MS2-NLS protein containing nuclear export sequences (NESs) of different origins. The NLS alone causes nucleoplasmic accumulation of YFP–MS2 and the formation of a diffuse nuclear YFP background, which could mask the detection of

544


A RT I C L E S a

b MS2 probe (Cy3) RNA-FISH

24 min

30 min

84 min

156 min

54 min

96 min Transcription site relative intensity

78 min

42 min

270 min

1.0 0.8 0.6 0.4 0.2 0

½-Mini-Dys +intron

Merge + DNA

½-Mini-Dys

Merge + DNA

126 min Top cell Bottom cell

0

100

200

300

Time (min)

c MS2 probe (Cy3) RNA FISH

DNA

Merge

Merge + mRNP detection

½-Mini-Dys +intron 8 h PonA

½-Mini-Dys

d

e

mRNP detection Nuclear mRNPs Cytoplasmic mRNPs

g 3

0

NES2

1

NES1

2

30 min

Untransfected

Average C/N ratio

f

120 min

60 min

180 min

½-Mini-Dys +intron

3

½-Mini-Dys + intron ½-Mini-Dys

2 1 0

½-Mini-Dys 90 min

240 min

h mRNP numbers

½-Mini-Dys +intron

4

Average C/N ratio

Nuclear mRNPs Cytoplasmic mRNPs

2

5

Time (h)

8

14

1,400 1,200

Cytoplasm Nucleoplasm

1,000 800 600 400 200 0

20

40

60

80 100 120 140 160 180 200 220 240 260 280

Time (min)

Figure 2 Quantification of mRNA nucleocytoplasmic transport. (a) Frames from a movie of a ½-Mini-Dys cell expressing NES1-YFP–MS2-NLS during active transcription and mRNP transport (Supplementary Information, Movie S1). Green and blue arrowheads indicate the induced transcription sites (times are after addition of PonA). Plot shows the time frame of transcription induction in both cells, which initiates at different time points. (b) RNAFISH comparing the cellular distribution of the ½-Mini-Dys mRNAs (red) either with (top) or without (bottom) an intron, after 6 h of induction by PonA. Hoechst DNA stain is in blue. (c) Measuring the mRNA C/N ratio: RNA-FISH with a probe against the MS2 region in the mRNAs (green) was imaged in three dimensions and deconvolved; mRNPs were detected in the nucleoplasm (red balls) and cytoplasm (white balls). Hoechst DNA staining is in blue. ½-Mini-Dys+intron (top) versus ½-Mini-Dys without an intron (bottom), after 8 h with PonA. (d) The detected mRNPs in the nucleus (red balls) and cytoplasm (white balls) in both cells. (e) RNA-FISH was performed at different time points after PonA activation, and the C/N ratios of the

quantified mRNPs in both cell types were calculated. This showed a high C/N ratio for the intron-containing transcripts at all time points (green), indicative of efficient export. Lower C/N ratios were seen for the intronless mRNA (pink), implying higher nuclear retention. At later times (14 h), once transcription had ceased, this retention was alleviated. Values are means ± s.d. measured for the two clones at the indicated times (2 h, ½-Mini-Dys+intron n = 4, ½-Mini-Dys n = 4; 5 h, ½-Mini-Dys+intron n = 4, ½-Mini-Dys n = 4; 8 h, ½-Mini-Dys+intron n = 3, ½-Mini-Dys n = 4; 14 h, ½-Mini-Dys+intron n = 4, ½-Mini-Dys n = 5 cells). (f) The NES1-YFP–MS2-NLS (NES1, n = 4 cells) and NES2-YFP–MS2 (NES2, n = 4) proteins did not affect C/N ratios of mRNAs, in comparison with untreated cells (n = 4), quantified by RNA-FISH. Values are means ± s.d. (g) Quantification of nuclear (red) and cytoplasmic (white) mRNPs in a 280 min time-lapse movie of a ½-Mini-Dys+intron cell. Blue, nuclear protein for detection of nuclear volume. (h) Bars show the quantified mRNPs in both compartments (yellow, nucleus; blue, cytoplasm) of the movie analysed in g over time. Scale bars, 10 µm.


545

A RT I C L E S Table 1 Summary of the measured diffusion coefficients for the various mRNAs in the nucleus and the cytoplasm mRNA transcript

Average Dnuc (µm2 s−1)

Maximum Dnuc (µm2 s−1)

Average Dcyt (µm2 s−1)

Maximum Dcyt (µm2 s−1)

Calculated tnc (min)

Measured tnc from live-cell movies (min)

E1

0.023

0.057

0.022

0.045

3.0–7.7

5

E3

0.010

0.024

0.017

0.037

6.9–17.6

20 15–20

E6

0.009

0.016

0.011

0.023

8.1–20.6

½-Mini-Dys

0.004

0.013

0.010

0.027

16.8–43.1

30


0.005

0.012

0.006

0.020

14.8–37.9

30

Mini-Dys

0.005

0.011

0.008

0.014

13.2–33.7

30–40

Full Dys

0.005

0.012

0.007

0.016

13.9–35.6

30–40

The average Dnuc values were used to calculate the travel time through a nucleus with a radius of 5–8 µm. Dnuc, nuclear diffusion coefficient; Dcyt, cytoplasmic diffusion coefficient; tnc, nucleocytoplasmic translocation time.

the initial time points of transcription. Both NES-containing versions were distributed evenly between the nucleoplasm and cytoplasm, thus providing an improved signal. The NES1 version contained the REVNES, whereas NES2 contained the heterogeneous nuclear ribonucleoprotein particle (hnRNP) A1 NES shuttling domain that is independent of Crm1 (ref. 28) (Supplementary Information, Fig. S5a). All three versions showed similar mRNP translocation kinetics (Supplementary Information, Fig. S5b, c), as analysed below. By using long-term livecell imaging followed by deconvolution we could detect transcription induction and the subsequent mRNA transport for all the genes (Fig. 2a; Supplementary Information, Movie S1). mRNP nucleocytoplasmic transport occurs over a 5–40 minute time range The Dys mRNPs were detectable in the nucleoplasm less than 10 min after the first time point of transcription site induction (about 3–6 min of elongation at about 1.5  kb min−1) and then filled the whole nucleoplasm (Fig. 2a). After another roughly 20–30 min, mRNPs were detectable in the cytoplasm and continued to accumulate there, indicating mRNA export. In addition, it was possible to follow the accumulation of fluorescent Dys protein hours later after induction (Supplementary Information, Movie S2). The global transport and export time frames, as observed by live-cell imaging, were similar for all Dys constructs. However, RNA-FISH showed that the intronless ½-Mini-Dys transcripts accumulated in the nucleoplasm over time: their levels increased (Fig. 2b) and they were retained probably as a result of lack of splicing, as previously suggested29. We counted the numbers of nucleoplasmic (N) and cytoplasmic (C) mRNA molecules at different time points after PonA induction and described export efficiency by calculating the C/N mRNA ratio. This analysis showed that the spliced ½-MiniDys+intron transcripts were efficiently exported even at long expression times, whereas the C/N ratio of the intronless transcripts was elevated when transcriptional activity was decreased (Fig. 2c–e). To demonstrate mRNP cytoplasmic accumulation in living cells we verified that the NES versions did not modify the C/N ratios (Fig. 2f). The number of YFP–MS2-labelled mRNPs accumulating in the nucleoplasm and cytoplasm were quantified over time, and showed a relatively constant number of nuclear mRNPs (about 400–450), in parallel with an increase in the cytoplasmic population (more than 1,000 mRNPs at 200 min; Fig. 2g, h). This single-cell analysis shows that the kinetics of the nuclear phase of gene expression (transcription, transport and export) entails a 5–40 min time frame (summarized in the right-hand column of Table 1). 546

The effects of RNA size and RNA splicing on nucleoplasmic diffusion rates To investigate mRNP export time in the context of total nucleoplasmic transport kinetics, we first quantified the nucleoplasmic phase of mRNP translocation. Nuclear transport of single Dys-mRNPs was quantified by live-cell imaging and two-dimensional or three-dimensional single-particle tracking. Tracked data were analysed by mean square displacement (MSD) analysis30, which is typically used to quantify the movement kinetics of the different mRNPs. In all cases, mRNP mobility measured in two (or three) dimensions followed a simple diffusion model, namely  = 4Dt, and portrayed either regular diffusion or movement typical of corralled diffusion, as described previously 24 (Fig. 3a–d; Supplementary Information, Movie S3). All Dys-mRNPs had relatively slow diffusion kinetics in comparison with previous measurements using the same tagging system in which a spliced 2.4-kb mRNA was analysed24. A wide range of Dys-mRNP nucleoplasmic diffusion rates were measured, ranging on an average D of 0.004–0.006 µm2 s−1 (Fig. 3e and Table 1). Three-dimensional tracking provided the same range of diffusion coefficients (Fig. 3f). Statistical analysis (see Methods) showed no significant differences between the diffusion rates of the different-sized Dys mRNAs. A small increase in the percentage of mRNPs presenting corralled diffusion was seen for the larger mRNPs (Fig. 3d). Nucleoplasmic travel times were calculated for all mRNAs using the above-measured average D values in a diffusion model of a three-dimensional volume ( = 6Dt) (nuclear radius = 5–8 µm)31 (Table 1). These calculations were compatible with the measured bulk nucleocytoplasmic translocation times from time-lapse movies (Table 1). These results demonstrate that the large size-differences in the larger mRNAs did not markedly change the nucleoplasmic diffusion coefficients and travel times (20–30 min), which could imply that these different-sized mRNAs were packed into similar large mRNP particles. Diffusion rates of the Dys-mRNPs in the cytoplasm were significantly faster than nucleoplasmic mRNP dynamics (Fig. 3e and Table 1). For further comparison of bulk mRNA export times and diffusion properties of other spliced and unspliced different-sized mRNAs, we included another set of genes in our analysis. These transcribed smaller mature mRNAs (1.7, 2.1 and 2.3 kb) that were generated from similar genes containing one, three and six exons, termed E1, E3 and E6, respectively (Fig. 4a, b). These YFP–MS2-labelled mRNPs appeared in the cytoplasm 5–20 min after transcription site induction (Table 1). The E1, E3 and E6 mRNPs showed diffusive and corralled movement and had higher diffusion coefficients (Fig. 4c, d). The latter were compatible with the measured bulk nucleocytoplasmic translocation times in living nature cell biology VOLUME 12 | NUMBER 6 | JUNE 2010

© 2010 Macmillan Publishers Limited. All rights reserved.

A RT I C L E S a

b

c

5

0.8 0.6

40 11 ss

50 s

62 s

0.4

3

MSD (µm2)

1s

y (µm)

4

2 1 0

85 s

69 s

96 s

1

2

3 4 x (µm)

60 40 20 0 ½-Mini

½-Mini + int.

Mini-Dys

Full Dys

5

f 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0

0

2

4

0

2

4

6

8

10

6

8

10

0.10

6

0.02 0

t (s)

E3D SPT

Nuclear Cytoplasmic

Y Z X

D (µm2 s–1)

80

Diffusive Corralled D (µm2 s–1)

Percentage of tracked mRNPs

0

e 100

0

0.06

102 s

d

0.2

½-Mini

½-Mini + int.

Mini-Dys

Full Dys

0.012 0.010 0.008 0.006 0.004 0.002 0

2D 3D


Figure 3 Analysis of mRNP kinetics. (a) Frames of a diffusive nucleoplasmic mRNP from a ½-Mini-Dys+intron cell tracked for 102 s (green track). (b) The full tracked movement from a (red, start of track; blue, end of track) (Supplementary Information, Movie S3). (c) MSD analysis of two tracked nucleoplasmic mRNPs, demonstrating diffusive (green) or corralled (red) movement. (d) Plot of the percentage of mRNPs showing either diffusive (blue) or corralled (red) motion for the four Dys genes in the nucleus. Values are means ± s.d. measured in independent experiments and in different cells of each clone. (e) Calculated average diffusion coefficients for nucleoplasmic

(purple) versus cytoplasmic (magenta) mRNPs. Values are means ± s.d. measured for each gene in the different compartments (nuclear, ½-MiniDys+intron n = 61, ½-Mini-Dys n = 38, Mini-Dys n = 48, full Dys n = 73; Cytoplasmic, ½-Mini-Dys+intron n = 55, ½-Mini-Dys n = 55, Mini-Dys n = 12, full Dys n = 49 mRNPs). (f) ½-Mini-Dys+intron mRNPs tracks (red) in three dimensions (n = 14 mRNPs) and plot of the measured diffusion coefficients compared with two dimensions (n = 47 mRNPs), showing a similar range of D values. 2D, two-dimensional; 3D, three-dimensional; SPT, single-particle tracking. Values are means ± s.d. Scale bars, 1 µm.

cells (Table 1). The mobility analysis of all the above transcripts shows that mRNP size influences the kinetics of the nuclear phase of mRNP translocation. The data for the large Dys-mRNPs suggest that the interchromatin sieve size may influence mRNP mobility, as recently shown for chromatin-devoid nuclei in C. tentans 32. We examine this below for the mammalian nucleus. In addition, we show that the splicing enhancement of export efficiency is not mediated through the dynamics of nucleoplasmic travels, because nuclear mRNP diffusion kinetics and the nucleocytoplasmic transport times were not enhanced for the intron-containing transcripts.

white region). We then searched for mRNP tracks traversing the nuclear envelope region (Fig. 5g, h), thereby allowing the analysis of potential export events. We detected mRNPs moving from the nucleoplasm into the cytoplasm (Fig. 5i; Supplementary Information, Fig. S6a, Movies S4 and S5). On the nuclear side, before export, mRNPs presented a spherical structure, whereas after export on the cytoplasmic side a disorganized open structure was seen, accompanied by a decrease in the YFP–MS2 signal on the mRNP, suggestive of restructuring and unfolding during export. Soon afterwards, mRNPs regained their rounded structure. (Only the 3´-tail fluorescent MS2 portion of the mRNP is detectable. See Supplementary Information, mRNP restructuring.) Similar results were obtained when the NPCs were marked by POM121-mCherry labelling (Supplementary Information, Fig. S6b, Movie S6). The measured time frame for single mRNP translocation through the NPC, on the basis of the imaging time resolution, was about 1 s. From the single-particle tracking data we calculated an average translocation velocity of 0.65 ± 0.37 µm s−1 (Fig. 5j, inset), thereby estimating an average time range of about 0.5 s or faster for translocation through the known pore size. A comparison of the MSD of diffusing nucleoplasmic or cytoplasmic mRNPs with the single mRNP export events (Fig. 5j) showed that mRNP movement through the pore was 15-fold faster than simple diffusion. This suggests that mRNP NPC-export is facilitated and rapid, and not based solely on diffusion33.

mRNA export is rapid and faster than nucleoplasmic diffusion To address the tracking of Dys-mRNPs during NPC export we used the YFP–MS2-NLS protein (without NES) as a marker for the inner nuclear boundary. We did not mark the NPCs to avoid tampering with the export machinery. We generated a maximum time projection of the acquired time-lapse movies, which means that the pixels of maximum intensity from each frame are visualized in one frame encompassing the whole movie. Using maximum time projections in the YFP–MS2 channel (Fig. 5a) we obtained clear border lines for the nucleus (Fig. 5b), because this protein does not stain the nuclear envelope. An average time projection (averaging the same pixels in all the frames of the complete movie sequence) allowed the detection of static mRNPs (Fig. 5c) in relation to mobile mRNPs, and showed that most mRNPs were mobile. Cyan fluorescent protein (CFP)–ZBP1 was used as a marker for the cytoplasmic boundary (Fig. 5d). With this time-projection procedure (Fig. 5e, f) we could define the nuclear envelope regions (Fig. 5g, arrowheads pointing to

mRNP NPC anchoring is independent of export Export proceeded rapidly once attachment of export-competent mRNP to the nuclear envelope was achieved, and no pile-up of mRNPs at the NPCs was observed (Supplementary Information, Movie S5). Static


547

A RT I C L E S a

E

YFP–MS2

E1 TRE + CMV min. prom.

Exon 1 + CFP

E1

E3

E6

3′ UTR

MS2 repeats

06SUREH&\


1

2

1

2

Exon 3 + CFP

MS2 repeats


4

5

Exon 6 + CFP

MS2 repeats

',&

d

'LIIXVLYH &RUUDOOHG

80

Nuclear Cytoplasmic

0.030

60 40

0.025 0.020 0.015 0.010

20 0

0.04 0.035

D (µm2 s–1)

3HUFHQWDJHRIWUDFNHGP513V

c 100

3

0.005 E1

E3

E6

½-Mini

½-Mini + int.

MiniDys

Full Dys

0

E1

E3

E6

½-Mini

½-Mini + int.

MiniDys

Full Dys

Figure 4 Measurements of nucleocytoplasmic dynamics of different mRNPs. (a) Three additional constructs were analysed. They all contain the Tetresponsive element (TRE) minimal cytomegalovirus (CMV) promoter (min. prom.) (grey), a CFP–SKL fusion protein (pink) fused to one exon (E1), three exons (E3) or six exons (E6). All contain MS2 sequence-repeats (yellow). (b) RNA-FISH to the MS2 regions showing transcription sites (white arrowheads) and cellular mRNAs (green). Differential interference contrast (DIC) is shown at the bottom. Scale bar, 10 µm. (c) Percentage of mRNPs presenting

diffusive (red) and corralled (green) movement types in the different cell clones (½-Mini-Dys+intron n = 4; ½-Mini-Dys n = 5; Mini-Dys n = 3; full Dys n = 4; E1 n = 3; E3 n = 3; E6 n = 3 cells). (d) The measured diffusion coefficients of all mRNAs in the nucleus (blue) (½-Mini-Dys+intron n = 61; ½-Mini-Dys n = 38; Mini-Dys n = 48; full Dys n = 73; E3 n = 69; E6 n = 58; E1 n = 61 mRNPs) and cytoplasm (yellow) (½-Mini-Dys n = 55; ½-MiniDys+intron n = 55; Mini-Dys n = 12; full Dys n = 49; E6 n = 25; E3 n = 33; E1 n = 39 mRNPs). Values are means ± s.d.

mRNPs were occasionally detected at the nuclear envelope. We then examined whether mRNP anchoring at occupied or unavailable NPCs could occur independently of mRNA export. Export was inhibited by treatment with wheat germ agglutinin (WGA)34 (see Supplementary Information), and a moderate increase in static mRNPs in the nuclear periphery was observed (Fig. 5k). mRNPs could be tracked engaging with the nuclear envelope (Fig. 5l; Supplementary Information, Movie S7) and remained anchored for relatively long periods (25–40 s). Occasionally, anchored mRNPs would detach after 2–12 s. Nucleoplasmic and cytoplasmic mRNP dynamics were unperturbed. These observations demonstrate that mRNA–NPC interactions are independent of export per se.

Information, Movie S8). In addition, the above time-projection analysis showing that most of the mRNP population was mobile (Fig. 5b, c) and that only few mRNPs were static, also demonstrated that mRNPs did not enter nucleolar regions (RNA-FISH and movies) (Fig. 6a–d, i–l; Supplementary Information, Movie S9). We assumed that restricted diffusion of mRNPs within inter-chromatin channels would be more efficient than movement in an open space, because of a decrease in dimensionality and because less nucleoplasmic space must be sampled. To test this, the channelled topography of the inter-chromatin space was disrupted by briefly shifting the cells to hyper-osmolar medium, which markedly and rapidly enlarges the inter-chromatin space and condenses chromatin, thereby forming interconnected relatively large corrals38 (Fig. 7a, b). Under these conditions there was no significant change in mRNP diffusion coefficients measured in the same cells before and after treatment (Fig. 7c). However, mRNPs sampled large inter-chromatin spaces in which they were corralled, as observed by an increase in the percentage of the corralled mRNP population (Fig. 7d–f). Furthermore, mRNPs had access to previously restricted nucleolar regions (Fig. 7a, b). Analysis of anomalous diffusion in the mRNP tracking data was negative, suggesting that the mRNPs were not interacting significantly with the channels in which they were moving 39. This analysis illustrates the functionality of chromatin in efficiently channelling mRNP transport towards the nuclear periphery as well as restricting entry into regions such as the nucleolus.

Channelled diffusion drives the mRNP nucleocytoplasmic flux The gene gating hypothesis envisioned a “space subjacent to the NPC and extending into the interior of the nucleus in the form of channels”35. The time-projection analysis allowed us to assess the nucleoplasmic tracks used by mRNPs (distinguishable from the free YFP–MS2 signal; Supplementary Information, Fig. S7). Time projections showed nucleoplasmic paths repeatedly used by different mRNPs (Fig. 6a–d), reminiscent of nuclear channels described previously 36,37. Three-dimensional imaging, together with DNA staining with Hoechst in living cells, showed mRNPs travelling within pathways intertwined between compact chromatin domains and in channels that reach the nuclear envelope (Fig. 6e–h; Supplementary 548


A RT I C L E S a

b

mRNPs

d

Av. Proj.

e

f Inverted max. proj.

Merge

CFP–ZBP1

g

c

Max. Proj.

h 18

&
 0.05). 50. Darzacq, X. et al. Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells. J. Cell Biol. 173, 207–218 (2006). 51. Tsukamoto, T. et al. Visualization of gene activity in living cells. Nature Cell Biol. 2, 871–878 (2000). 52. Dultz, E. et al. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J. Cell Biol. 180, 857–865 (2008). 53. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998). 54. Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nature Struct. Mol. Biol. 14, 796–806 (2007).

nature cell biology


s u p p l e m e n ta r y i n f o r m at i o n

Supplemental Figure S1

DOI: 10.1038/ncb2056 a

b GFP

anti-Dys

DIC

Full-Dys Cerulean

Mini-Dys

½-Mini-Dys

½-Mini +intron

Figure S1 Complete Control Inducible Mammalian Expression System. (a) Regulation of transcription in the Complete Control system (Stratagene). VgEcR and RXR bind as a heterodimer to form the nuclear receptor that binds to the E/GRE recognition sequence. In the absence of PonA (the inducer), the promoter is tightly repressed by corepressors. When PonA binds to VgEcR, the corepressors are

released, coactivators are recruited, and the complex becomes transcriptionally active. The pERV3 and pEGSH vectors are shown. (b) Dystrophin production in the 4 clones after PonA induction, detected by the fluorescent fusion protein (GFP or Cerulean; green or cyan) and immunofluorescence using an anti-dystrophin antibody (red). DIC is in the right panels. Scale bar, 20 µm.

www.nature.com/naturecellbiology

1 © 2010 Macmillan Publishers Limited. All rights reserved.


Supplemental Figure S2 a

b 1

2

3

2000bp 1500bp

c

t

t a

a

g

c

t

t a

a

g

Arbitrary RNA levels

c

9 8 7 6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

Time (hr)

Figure S2 Correct splicing of ½-Mini-Dys+intron. (a) RT-PCR on cDNA from DNase-treated RNA from the ½-Mini-Dys+intron cell line (lane 2), and on the original ½-Mini-Dys+intron plasmid (lane 3), with primers that span the splice site. Bands of the expected size are seen. Lane 1 = marker. (b) The spliced product was sequenced and showed the correct sequence expected after splicing, which contains two tandem AflII restriction sites

2

(CTTAAG-CTTAAG). (c) Fold induction of ½-Mini-Dys+intron mRNA over time following PonA induction, was quantified using semi-quantitative RTPCR in comparison to GAPDH mRNA levels. This showed that from 4 hrs and onwards cells should contain sufficient mRNA numbers, to be useful for live-cell imaging experiments. Values are mean ± s.d. measured in 3 independent experiments.

www.nature.com/naturecellbiology © 2010 Macmillan Publishers Limited. All rights reserved.


Supplemental Figure S3 MS2-Cy3 probe

DNA

Merge

DIC

30 min PonA

1 hr PonA

2 hr PonA

4 hr PonA

6 hr PonA

Figure S3 RNA-FISH of the ½-Mini-Dys+intron transcripts after different activation times with PonA, using the MS2-Cy3 probe. Transcription sites

(large red dot) and cellular mRNAs (red dots) are seen. Hoecsht DNA stain is in blue, and DIC is in the right panels. Scale bar, 10 μm.




Supplemental Figure S4 MS2-Cy3 probe

DNA

Merge

DIC

30 min PonA

1 hr PonA

2 hr PonA

4 hr PonA

6 hr PonA

Figure S4 RNA-FISH of the ½-Mini-Dys (-intron) transcripts after different activation times with PonA, using the MS2-Cy3 probe. Transcription sites

4

(large red dots) and cellular mRNAs (red dots) are seen. Hoecsht DNA stain is in blue, and DIC is in the right panels. Scale bar, 5 µm.



Supplemental Figure S5 a Untreated

+LMB

YFP-MS2-NLS

YFP-MS2-NLS

DNA

NES1-YFP-MS2-NLS

NES1-YFP-MS2-NLS

NES2-YFP-MS2

NES2-YFP-MS2

b 0

DNA

c 10min

20min

0.01 NLS NES2

0.008

NES1 40min

50min 0.006

2

D (µm /s)

30min

0.004 60min

70min

80min 0.002

0

Figure S5 NES-YFP-MS2-NLS proteins do not modify mRNPs kinetics. (a) Subcellular distribution of transiently transfected YFP-MS2-NLS (top), NES1-YFP-MS2-NLS (REV-NES, middle), and NES2-YFP-MS2 (hnRNP A1 NES/NLS, bottom) proteins. Treatment with leptomycin B (LMB) shows that NES1 and not NES2 is Crm1-dependent. Scale bar, 20 µm. Hoechst DNA staining in blue. (b) Frames from a movie of a ½-Mini-Dys+intron cell expressing YFP-MS2-NLS (green) during active transcription and mRNP

½-Mini-Dys +int

transport, demonstrating similar transport kinetics as measured with mRNPs tagged with NES1-YFP-MS2-NLS (Fig. 2a). Arrow points to the induced transcription site (green dot). CFP-ZBP1 was used as a cytoplasmic marker (pseudo-colored red). Scale bar, 5 µm. (c) The diffusion coefficients measured by SPT of ½-Mini-Dys+intron mRNPs were in the same range when tagged by either YFP-MS2-NLS (n=19 mRNPs), or NES1-YFP-MS2NLS (n=42), or NES2-YFP-MS2 (n=23). Values are mean ± s.d.




Supplemental Figure S6 a

b

0

3s

6s

0

1s

3s

6s

1s

4s

7s

2s

5s

7s

Figure S6 Live-cell imaging of mRNP export. (a) Frames showing single mRNPs before, during and after export (Movie S5). The nuclear membrane is marked with a dashed line. mRNP is seen in yellow (Fire lookup table).

6

2s

0

0.4s

0.8s

5s

1.2s

1.6s

2s

Max

2.4s

2.8s

Max

(b) Frames showing a single mRNP before, during and after export (Movie S6). The nuclear membrane is marked by co-transfection with mCherry-pom121 (Red). Max = maximum projection (Fire lookup table). Scale bar, 1 µm.



Supplemental Figure S7 YFP-MS2 No RNA

1 sec

5 sec

10 sec

Max. Proj.

DIC

YFP-MS2 with RNA

Figure S7 Free YFP-MS2 proteins do not interfere with the detection of mRNP nuclear tracks. Naive U2OS cells (top, no RNA) and a cell from the ½-Mini-Dys+intron clone (bottom) were transfected with YFP-MS2-NLS.

After PonA induction, cells were imaged every 1 sec. Representative frames from t= 1, 5 and 10 sec are shown. Max projection of the total time frames are marked as Max. Proj. DIC is in the right panels. Scale bar, 5 µm.




Supplementary Movie Legends Movie S1 Transcriptional induction and distribution of mRNPs in living cells. Pon A was added to ½-Mini-Dys-(-intron) cells at the beginning of the movie and images were acquired in 3D every 6 min. The movie runs for 6 hr and 42 min, and shows the induction of transcription sites, the appearance of nucleoplasmic mRNPs, followed by accumulation of cytoplasmic mRNPs. The free YFP-MS2 (yellow) is found throughout the whole cell (and nucleus) in the beginning of the movie. It then binds to the many mRNPs generated by the transcription site, and towards the end of the movie only real mRNPs are labeled thereby reducing the background signal to a bear minimum. The transcription sites continue to transcribe but makes less RNAs. Movie S2 Accumulation of mRNA and ½-Mini-Dys protein product. Left- YFP-MS2 labeled mRNA (yellow). Right – ½-Mini-Dys protein (cyan). PonA was added to ½-Mini-Dys+intron cells 15 min prior to imaging. Cells are seen dividing and then transcription sites are induced followed by accumulation of the ½-MiniDys protein. Some non-specific Cerulean signal can be seen over the region of the transcription site. Images were acquired in 3D every 20 min, for 15 hrs. Movie S3 Tracking of a diffusing mRNP. A YFP-MS2 labeled nucleoplasmic mRNP from a ½-Mini-Dys+intron cell. Imaged every 1 sec for 109 sec. Green annotates the measured track. Note the top mRNP that does not move throughout the movie. Movie S4 Single mRNP nuclear export (1). A nuclear mRNP (green) in a ½-Mini-Dys+intron cell is observed as it translocates into the cytoplasm (blue). Imaged every 1 sec for 8 sec. The middle movie includes the track in red and the right-hand panel shows the signal-only in yellow (pseudo-colored using fire look-up table). Movie S5 Single mRNP nuclear export (2). A nuclear mRNP (white) in a ½-Mini-Dys+intron cell is observed as it translocates into the cytoplasm. Nuclear boundaries are demarcated in red. Also seen are the transcription site (large white locus), and at the bottom note an mRNP that does not translocate. Imaged every 1 sec for 10 sec. Left and right are the same movie. The right-hand panel includes the track in green. Movie S6 Single mRNP nuclear export (3). A nuclear mRNP (green) in a ½-Mini-Dys cell is observed as it translocates into the cytoplasm. NPCs are marked by co-transfection with mCherry-pom121 (Red). Imaged every 0.4 sec for 3.6 sec. The right-hand image includes the track in blue. Movie S7 Single mRNP nuclear envelope anchoring. A nuclear mRNP in a WGA treated ½-Mini-Dys cell, is observed as it diffuses towards the nuclear envelope. When the mRNP reaches the envelope it abruptly anchors. Note that there is another anchored mRNP throughout the whole movie. Imaged every 1 sec for 23 sec. Movie S8 mRNPs are found in inter-chromatin channels. 3D-rendering of a 3D stack from a live cell containing mRNPs (green) and stained chromatin (Hoechst, pseudo-colored red). Movie S9 mRNPs are excluded from the nucleolus. YFP-MS2 labeled transcription site and mRNPs are seen in green in a ½-Mini-Dys+intron cell. The cytoplasm is in red and marked with RFP-α-tubulin. Nucleoplasmic mRNPs do not travel through the nucleoli (seen as black holes). Imaged every 1 sec for 71 sec. Movie S10 mRNPs are trapped in large corrals during osmolar treatment. A cell showing mRNPs (green) and Hoecsht DNA stain (red) before (left) and after (right) osmolar treatment. After treatment, large inter-chromatin spaces open up and mRNPs move within. Imaged every 1 sec for 60 sec.

8


Supplementary Information for Mor et al. Dynamics of single mRNP nucleo-cytoplasmic transport and export through the nuclear pore in living cells

Dystrophin gene Muscular dystrophin is a cytoskeletal protein found at the inner surface of muscle fibers. It is part of the dystrophin-glycoprotein complex (DGC), which bridges the inner cytoskeleton (F-actin) and the extra-cellular matrix (ECM). Mutations in DMD are typically truncating, removing the C-terminal half of the protein, thereby being unable to attach to the DGC and to the ECM. The dystrophin protein is the product of the gene defective in Duchenne muscular dystrophy (DMD), the largest gene found so far in nature. The DMD gene encodes a range of different transcripts, and a large set of protein isoforms are formed. The full-length 14 kb mRNA consisting of 79 exons that encodes the 427 kD dystrophin protein (11 kb ORF, 3685 amino acids) active in muscle. The GFP-dystrophin construct used herein was shown to encode a functional dystrophin protein1. Due to the large size of the gene there were no restriction sites left that would enable the changing of GFP with Cerulean as performed with the other Dys constructs. The mini-dystrophin variant used in this study encodes a protein of 210 kDa which lacks the spectrin-like domain2.


Complete Control Expression System The inducible transcription system is based on robust activation of transcription by ponasterone A (PonA) known as the Complete Control® Inducible Mammalian Expression System (Stratagene). PonA is an analog of the insect hormone ecdysone and in this system PonA is activating transcription in mammalian cells harboring both the gene for the Drosophila melanogaster ecdysone receptor (EcR) and a promoter containing a binding site for the ecdysone receptor. PonA has no measurable effect on mammalian physiology, has a short in vivo half-life, and its lipophilic nature allows it to efficiently penetrate all tissues. The expression system is based on two vectors that are required for the expression of the gene of interest: the pERV3 receptor vector and the pEGSH expression vector. The pERV3 vector contains an expression cassette from which the VgEcR and RXR proteins are constitutively expressed. VgEcR is a synthetic ecdysone-binding receptor which is a member of the retinoid-X-receptor (RXR) family of nuclear receptors. VgEcR heterodimerizes with RXR and together they regulate transcription. The pEGSH vector contains a PonA-inducible expression cassette that includes five copies of the E/GRE recognition sequence (a synthetic recognition site that does not bind any endogenous transcription factors) and a multiple cloning site (MCS) for inserting the gene of interest. Induction of the system (Supplementary Fig. S1a) occurs upon heterodimerization of VgEcR and RXR and the formation of the nuclear receptor that binds to the E/GRE elements upstream of the gene of interest. In the absence of PonA, co-repressors are bound to the receptor and repress transcription. When added, PonA binds to the receptor and the


co-repressors are released, consequently, permitting transcriptional activation (Supplementary Information, Fig. S1a).

mRNP restructuring What is the structure of a mature mRNP? Structural data is not available. The notion is that mRNPs travel as a spherical particle, and this is enforced by EM studies such as the Balbiani ring mRNPs3 and others4. In the export analysis presented, we examined whether restructuring could be visually detected. We are referring to the unfolding of the structure rather than restructuring by hnRNP component exchange on the mRNP prior to NPC passage. In our analysis, we detect on the cytoplasmic side the return from an “open” mRNP structure to a spherical structure. We suggest this to be indicative of restructuring occurring during export. Indeed, typical mRNP diameter is in the range of 20 nm, and the central channel of the pore has been recently recalculated and shown to be 2.6 nm5. This would mean that any mRNP must undergo some kind of "restructuring" in order to pass through. The nature of this restructuring has yet to be defined. It should be noted that the MS2 stem-loops are situated in the 3'UTR of the mRNAs analyzed, and therefore this 3' tail is the only detectable portion of the mRNA. Therefore, this portion might help in predicting what is occurring structurally with the rest of the mRNA molecule. However, the point at which restructuring begins cannot be defined with our image acquisition times. This means that the “open” structure observed prior to NPC translocation could be due the movement of the mRNP during imaging, or even due to


events occurring on the 5’-end of the mRNA which we cannot detect (the mRNA is thought to translocate 5’-end first through the NPC). We have estimated the size of the MS2 region as follows. The MS2 sequence is 1256 nt’s long. The physical length of the linear sequence would be 427 nm (each nucleotide 0.34 nm). However, the stem-loops (24 x 19 nts = 155 nm) are folded structures and therefore the length of the mRNA would be 272 nm (427-155), plus the width of each stem-loop (1.08 nm as in an A-T bond; 24 * 1.08 =26 nm). Therefore, the total length of an “open” MS2 region would be in the range of ~300 nm, which is in the limits of our light microscope resolution.

Estimation of mRNP nucelo-cytoplasmic travel times Previously measured diffusion coefficients of a 2.4 kb mRNA labeled with YFP-MS26 were later used to simulate the time of mRNP translocation to the cytoplasm7. This analysis suggested a 6 min translocation time for the 2.4kb mRNA, rather than the predicted second range. Using the diffusion equation MSD= 6Dt in 3D we could estimate the time required for an average mRNP to travel distances of 5-8 µm which are typical mammalian nuclear radii8. The measured diffusion coefficients were used to calculate the translocation times (t), and provided translocation times shown in Table 1. This time range was also measured in vivo in the live-cell movies, in which the appearance of mRNPs in the cytoplasm was timed in relation to the starting point of mRNP nuclear appearance (Fig. 2a and Table 1).


WGA treatment WGA is a lectin that binds N-acetyl glucosamine (GlcNac) groups found in some of the phenylalanine-glycine (FG) repeats of nucleoporins, particularly the central Nup62 complex9-12. Since WGA has high binding affinity to oligomeric GlcNac's, it is proposed that it introduces additional mesh contacts into the central channel of the NPC, thereby reducing its radius5, and inhibiting nucleo-cytoplasmic transport. WGA treatment requires digitonin permeabilization of the cytoplasmic membrane only. Conditions were calibrated for the lowest concentration of digitonin treatment that enabled the penetration of Cy5labeled WGA into the cells and cell survival for several hours. Both were monitored under the microscope on living cells. When WGA-Cy5 was added in the presence of digitonin a slow increase in mRNPs stalled on the nuclear membrane was observed. Since there are many species of nuclear mRNPs we expect to see only some of the YFP-MS2 labeled mRNPs stuck on the nuclear envelope.

1. 2. 3. 4. 5. 6.

Chapdelaine, P. et al. Functional EGFP-dystrophin fusion proteins for gene therapy vector development. Protein Eng 13, 611-615 (2000). England, S.B. et al. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 343, 180-182 (1990). Mehlin, H., Daneholt, B. & Skoglund, U. Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography. Cell 69, 605-613 (1992). Franke, W.W. & Scheer, U. Structures and functions of the nuclear envelope, in The Nucleus, Vol. 1. (ed. H. Busch) 219-347 (Academic Press, New York; 1974). Mohr, D., Frey, S., Fischer, T., Guttler, T. & Gorlich, D. Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J. 28, 2541-2553 (2009). Shav-Tal, Y. et al. Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797-1800 (2004).


7. 8. 9. 10. 11. 12.

Braga, J., Rino, J. & Carmo-Fonseca, M. Photobleaching microscopy reveals the dynamics of mRNA-binding proteins inside live cell nuclei. Prog Mol Subcell Biol 35, 119-134 (2004). Ribbeck, K. & Gorlich, D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J 20, 1320-1330 (2001). Nagata, Y. & Burger, M.M. Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J Biol Chem 249, 3116-3122 (1974). Finlay, D.R., Newmeyer, D.D., Price, T.M. & Forbes, D.J. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol 104, 189-200 (1987). Hanover, J.A., Cohen, C.K., Willingham, M.C. & Park, M.K. O-linked Nacetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J Biol Chem 262, 9887-9894 (1987). Davis, L.I. & Blobel, G. Identification and characterization of a nuclear pore complex protein. Cell 45, 699-709 (1986).
