Visualization of Single RNA Transcripts in Situ
Abstract
Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of β-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.
The identification of specific nucleic acid sequences by FISH has revealed sites of RNA processing, transport, and cytoplasmic localization (1). Recognition of these sites of hybridization is possible only when sufficient concentrations of the target sequence provide contrast with regions of lesser or no signal. Here we describe a quantitative approach to identify single molecules in these regions of low concentration. The methodology also facilitates accurate quantitation of the regions containing multiple copies of RNA, such as is found at transcription sites. Analysis of individual transcription sites with single molecule accuracy generated precise information on nascent chain initiation, elongation, and termination.
FISH images are composed of points of light with variable intensities resulting either from hybridization or from background fluorescent noise. We used multiple probes targeted specifically to β-actin mRNA to generate high-intensity point sources that result from hybridization to individual RNAs. We then quantitated the light intensity from each point source to distinguish hybridization events from spurious fluorescence.
The strategy involves (i) synthesizing several oligonucleotide probes to adjacent sequences on an RNA target such that their collective fluorescence will be emitted as a point source after hybridization; (ii) conjugating fluorochromes to specific sites on each oligonucleotide probe so that the fluorescent output per molecule of probe can be calibrated (Fig. 1, A to C); (iii) acquiring digital images from a series of focal planes through a hybridized cell; and (iv) processing these images with a constrained deconvolution algorithm such that out-of-focus light is quantitatively restored to its original points of origin.
To identify single β-actin mRNA molecules, we hybridized multiple probes to the isoform-specific 3′-untranslated region (UTR) of the mRNA in normal rat kidney (NRK) cells. The acquired fluorescence image was made up of numerous bright points of fluorescence interspersed within a diffuse, lower intensity signal (Fig. 1D). After deconvolution, the image consisted of numerous distinct points of light throughout the cytoplasm (Fig. 1E). The brightest points of light were constrained to a few contiguous volume elements (voxels). The image also contained scattered single voxels and clusters of voxels with low amounts of fluorescent signal attributed to low-level autofluorescence and sources of background noise. Establishing an image threshold removed ∼95% of these voxels (Fig. 1, F and G). The discrete, brightest clusters that remained after a threshold was established are referred to as objects. The total fluorescence intensity (TFI) of each object was then calculated. These bright objects had TFI values expected for single molecules and fell within the range of one to five probes hybridized (Fig. 1H). This analysis supports the conclusion that single molecules of β-actin mRNA were being detected and that they corresponded to the points of light. The β-actin mRNA molecules were often far enough apart to be resolved by light microscopy as individual point sources.
Additional evidence that these points of light are single molecules comes from an analysis of their prevalence. A population of cells grown in serum-free medium contained 500 ± 200 β-actin mRNAs per cell. In an exponentially growing population of cells the number increased to ∼1500 copies, consistent with activation of actin mRNA transcription and possible stabilization of cytoplasmic actin mRNA by serum (2, 3). This copy number of β-actin mRNA agrees with estimates of the average abundance of β-actin mRNA per cell derived from molecular techniques (4).
Finally, spectrally distinct probes hybridized to different target sequences on the same mRNA molecule were used to verify single molecule detection. When the probes were targeted to the same molecule, the signals were close together (Fig. 2C). The TFI confirmed that they were hybridized to only one molecule. Statistical analysis of 478 signals in the cell determined that 59% of the red signals were ≤3 voxels (279 nm) from a green signal. As a control, two isoforms of actin mRNA, β and γ, were detected simultaneously in the same cell with probes that were specific for each isoform (Fig. 2, A and B). The gene sites for each isoform were distinguishable in the nucleus and the mRNAs as separate point sources in the cytoplasm. In contrast to the cis probes, the signals did not colocalize significantly; they were ≤3 voxels apart 13% of the time.
Visualization and measurement of intramolecular distances were possible when the cis probes were sufficiently separated (Fig. 2, C and D). To ascertain that the displacement of the hybridization signal was not an artifact due to misalignment of the respective red and green images, we hybridized three probes, each labeled with distinct fluorochromes, to β-actin mRNA at distances of 1648 nucleotides (nt) (maximum) and 631 nt (minimum) apart. We found that the registered images from the maximally distant probes were not coincident (as much as 487 nm apart), whereas near probes were within the same voxel. This result indicated that the detected mRNA was almost completely extended (1648 kb × 0.3 nm per nucleotide = 494 nm).
To verify that a single copy sequence could be detected by this method, we hybridized 10 probes, each with six CY3 fluorochromes, to the template strand of the β-actin gene (Fig.3A). Eleven probes, each with five fluorescein isothiocyanate (FITC) fluorochromes, targeted to the β-actin nascent transcripts were used simultaneously (Fig. 3B) and verified the gene site by colocalization (Fig. 3C). The TFI values obtained for hybridization to the single DNA target (60 fluorochromes) were consistent with the TFI predicted from single mRNAs. The nascent transcripts gave a much brighter signal than the gene because they represented multiple RNA molecules concentrated at that site. Measurement of 130 genes revealed the distribution of fluorescence expected from the independent hybridization of 10 probes (Fig. 3D). The hybridization efficiency was close to 100%, because all 10 probes hybridized most frequently. Hybridization to nascent transcripts indicated the presence of multiple copies corresponding to a maximum of 68 probes per transcription site.
This technology allowed us to quantitate the kinetics of β-actin mRNA transcription (Fig. 4). Cells cultured overnight in serum-free medium showed essentially no detectable transcriptional activity (Fig.4A). The addition of serum (2) resulted in synchronous activation of transcription detected on one of the β-actin alleles in virtually every cell within a few minutes (Fig. 4B). The number of nascent transcripts per β-actin allele was determined with a single probe to the 5′-UTR. The simultaneous use of 3′-UTR probes provided information on the number of polymerase molecules that had progressed to the terminal portion of the gene. At 3 min after induction, the average gene contained 12 nascent RNAs, but few 3′-UTR signals (Fig. 4, B and G). By 4 min, both β-actin alleles were transcriptionally active in almost all cells, and four transcripts had entered into the 3′-UTR (Fig. 4G). These results are consistent with a transcription rate of 1.1 to 1.4 kb/min (5). The mean number of nascent transcripts associated with each gene increased linearly from 0 to 5 min, with a mean increase of four transcripts per minute (Fig. 4H). Few transcripts dissociated from the site during this 5-min interval, as evidenced by the probe fluorescence constrained to the location of the gene (Fig. 4C).
Between 5 and 15 min after induction, the number of nascent RNA transcripts per allele increased steadily and peaked at 15 min, with an average of 30 nascent transcripts per allele (Fig. 4H). During the same period, the mean number of transcripts traversing the 3′-UTR reached a steady-state value of 8 to 10, indicating that the rate of entry of new transcripts into this region was equal to the rate of transcripts terminating. At this time, spots of fluorescence were detected around the transcription site (Fig. 4, D and E). These point sources of fluorescence were less intense than the transcription sites. Their TFI values were identical to the points of signal in the cytoplasm; therefore, we interpreted them to be single, terminated transcripts leaving the site of the gene.
Because the released transcripts did not accumulate significantly in the immediate vicinity of the gene after the expected time of termination, we concluded that the initiation of transport took no more than a few minutes. The dispersal of transcripts away from the transcription site took on a variety of spatial patterns (Fig. 4, D and E). Often tracklike distributions were apparent, and the regular spacing between points suggested a constant rate of movement away from the transcription site. Occasionally, the transcripts appeared to spiral away from the site of transcription; however, in at least half of the cases examined, the terminated transcripts simply appeared to diffuse around the site, without any discernible pattern. Nascent transcripts diminished at 60 min after serum induction (Fig. 4F). The decline of transcriptional activity beginning at 30 min fit an exponential decay profile for the next 1.5 hours, at which time it became indistinguishable from the state before induction (Fig. 4H). Over the induction period beginning at 10 min, there was a constant increase in the number of single β-actin mRNA molecules in the cytoplasm (6).
The capacity of these procedures to generate accurate information about single cells was illustrated by a high-resolution, dynamic view of each β-actin mRNA transcription site obtained with three probes positioned along the RNA, each labeled with a different fluorochrome (Fig. 4I). A “snapshot” of a representative gene revealed 23 nascent RNAs in the process of elongation, 14 of which had progressed through the proximal 3′-UTR and 8 of those through the distal 3′-UTR. At least five of the RNAs had passed the cleavage and polyadenylation site. These RNAs may be nascent, cleaved but undergoing polyadenylation, or awaiting transport. These possibilities should be distinguished by using probes downstream from the cleavage and polyadenylation site. The rat β-actin gene has a consensus “termination” sequence at the predicted distance downstream of the polyadenylation sequence that might account for this observation (7). The average spacing between polymerases in the 3′-UTR was ∼60 nt, whereas in the 5′-region of the gene, it was ∼170 nt. This spacing is consistent with previous observations on polymerase II (Pol II) transcription loading (8). The closer packing of polymerases in the 3′-UTR of the gene suggested that termination and release were rate limiting. Recent work indicates that Pol II recruits cleavage and polyadenylation factors, which may result in the polymerase slowing at this point (9).
The rate of increase (four transcripts per minute) in the number of nascent transcripts from 0 to 5 min reflects solely the rate of initiation, because few polymerases should have reached the termination site, on the basis of the rate of elongation. An in vitro system indicated that Pol II can initiate at a rate of two transcripts per minute (10). Between 5 and 15 min, when termination was occurring, there was a sustained increase of one nascent RNA per minute. Assuming that the initiation rate remained constant, then the rate of termination and release would be three RNA per minute. Surprisingly, there was a precipitous decrease of activity at the transcription site after 30 min. The cycle of the transcriptional pulse in response to serum is similar to that shown for c-fos(11). By interrogating the site with probes along the length of the RNA, we determined that this shut-down resulted from an inhibition of initiation rather than “pausing” of the polymerases. There was no apparent accumulation of transcripts at the nuclear envelope, so it is likely that export is not rate limiting.
The advance herein evaluates FISH images so that single molecule signals are not eliminated through background subtraction or the establishment of a threshold. As a consequence of this approach, single mRNA molecules were detectable with as little as one oligonucleotide probe, containing only five fluorochromes, an increase in sensitivity more than two orders of magnitude greater than previously obtained (12). In addition, the template DNA strand was accessible to the small probes during transcription without denaturation, contrary to other protocols, and may indicate that it contained single-stranded regions. This technique may therefore allow probing of the active regions of chromatin and facilitate detection of specific genes or groups of genes where only short, nonrepetitive sequences can be used. This would bring many physiologically important genes for receptors, signaling molecules, cell cycle regulators, and transcription factors, as well as their nuclear and cytoplasmic transcripts, into range of FISH detection.
Footnote
†
Deceased.
Supplementary Material
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REFERENCES AND NOTES
1
J. B. Lawrence, R. H. Singer, L. M. Marselle, Cell 57, 493 (1989); C. L. Sundell, R. H. Singer, Science 253, 1275 (1991); G. Zhang, K. L. Taneja, R. H. Singer, M. R. Green, Nature 372, 809 (1994); Long R. M., et al., Science 277, 383 (1997).
2
Elder P. K., Schmidt L. J., Ono T., Getz M. J., Proc. Natl. Acad. Sci. U.S.A.81, 7476 (1984).
3
Greenberg M. E., Hermanowski A. L., Ziff E. B., Mol. Cell. Biol.6, 1050 (1986).
4
J. B. Lawrence, R. H. Singer, Nucleic Acids Res. 13, 1777 (1985); Schwartz R. J., Rothblum K. N., Biochemistry 20, 412 (1981).
5
Thummel C. S., Science255, 39 (1992); A. W. Shermoen and P. Z. O'Farrell, Cell67, 303 (1991).
6
A. M. Femino, F. S. Fay, K. Fogarty, R. H. Singer, data not shown.
7
R. Ashfield et al., EMBO J. 13, 5656 (1994); R. Ashfield, P. Enriquez-Harris, N. Proudfoot, ibid. 10, 4197 (1991).
8
Osheim Y. N., Miller O. L., Beyer A. L., Cell43, 143 (1985).
9
Dantonel J.-C., Murthy K. G. K., Manley J. M., Tora L., Nature389, 399 (1997).
10
Hawley D. K., Roeder R. G., J. Biol. Chem.262, 3452 (1987).
11
Huang S., Spector D. L., Genes Dev.5, 2288 (1991).
12
R. W. Dirks, K. C. Daniel, A. K. Raap, J. Cell Sci. 108, 2565 (1995); Xing Y., Johnson C. V., Moen P. T., McNeil J. A., Lawrence J., J. Cell. Biol. 131, 1635 (1995).
13
For details of the digital imaging microscopy procedures, see F. S. Fay, K. E. Fogarty, J. M. Coggins, Soc. Gen. Physiol. Ser. 40, 51 (1986); Fay F. S., Carrington W. A., Fogarty K. E., J. Microsc. 153, 133 (1989). . Images were obtained with an inverted Nikon Diaphot 200 epifluorescence microscope equipped with a 100-W mercury lamp and modified to capture images under computer control. Between 20 to 35 optical sections were acquired at 250-nm z-intervals and at an effective pixel size of 187 nm by 187 nm with a thermoelectrically cooled (–45.0°C) charge-coupled device (CCD) (model 220; back-thinned RCA CCD chip; 50 kHz; Photometrics, Tucson, AZ). A high quantum efficiency (0.8 at 500 nm) and a low noise (50 photons equivalent) were required for imaging at the low light levels.
14
Oligonucleotide probes were synthesized, purified, and labeled as described [Kislauskis E. H., Li Z., Singer R. H., Taneja K. L., J. Cell Biol. 123, 165 (1993)] (23). Below are listed the probes used in this work (nucleotide number start, length in nucleotides). The sequence is for β-actin unless specified (accession J00691). For Fig. 1, B and D through H, the antisense probes to the 3′-UTR were 3133, 51; 3312, 52; 3434, 53; 3488, 52; and 3542, 52. For Fig. 2, same as Fig. 1 and the antisense probes to the γ-actin (accession X52815) 3′-UTR were 1151, 50; 1282, 61; 1409, 52; 1470, 54; and 1535, 56. For Fig. 2 C, same as Fig. 1 and the antisense probes to the splice junctions were (exon nucleotides only) 284, 52; 1340, 52; 1667, 52; 2567, 56; and 2839, 53. For Fig. 2 D, the antisense probe to the 5′-UTR was 255, 50; to the coding region, 2839, 53 and 3034, 52; and to the 3′-UTR, 3542, 52. For Fig. 3 A, the sense probes to the introns were 630, 63; 998, 63; 1069, 63; 1369, 63; 1691, 61; 1889, 64; 1960, 63; 2040, 63; 2590, 63; and 2864, 62. For Fig. 3 B the antisense probe to the 5′-UTR was 225, 53; to the splice junctions, the same as Fig. 2; and to the 3′-UTR, the same as Fig. 1 . For Fig. 4, A through F, the antisense probe to the 5′-UTR was 225, 53 and the antisense probes to the 3′-UTR were 3133, 51; 3312, 52; 3434, 53; 3488, 52; and 3542, 52. For Fig. 4 I, the antisense probe to the 5′-UTR was 255, 50, and the antisense probes to the 3′-UTR were 3133, 51 and 3542, 52.
15
The TFI per probe was measured by using a known concentration of fluorochrome in a determined volume between a cover slip and slide as follows: (i) Probes were dispersed in a solution equivalent in composition to mounting medium with final concentrations ranging from 0.25 to 4.0 ng/μl for cyanine (CY)3- and CY5-labeled probes and 1.0 to 16 ng/μl for FITC-labeled probes. (ii) Fluorescent beads (200 nm in diameter) adhered to the glass surfaces and delineated the distance between the inner surfaces of the cover slip and slide when brought into focus at each surface. The stage position was monitored with an Eddy-current sensor and was used to measure the vertical distance between the two beads to determine the height of the volume element containing the probe solution. (iii) A single plane in the middle was imaged and contained the total fluorescence contributed by all the fluorescent molecules in solution above and below the plane of focus in the imaged volume [imaged volume = (x)(y)(z) μm3]. The pixel size was calibrated for the optical setup of the microscope (x = y = 93 or 187 nm with a ×60 magnification, 1.4 numerical aperture objective, and ×5 or ×2.5 camera eyepiece, respectively). The TFI is dependent on the light flux at the sample; therefore, a calibration curve was calculated for each imaging session. The slope is equal to the TFI per fluorochrome under imaging conditions (CY3: m = 65 ± 1.3,R = 0.992; FITC: m = 20.6 ± 0.8,R = 0.985; CY5: m = 56.3 ± 1.4; correlation coefficient R = 0.992). Exposure times for CY3, FITC, and CY5 were 3, 4, and 15 s, respectively. (iv) The TFI value per probe obtained from solution was adjusted to that for a restored image and multiplied by a factor (50) equal to the number of optical sections in the point spread function (PSF) used for deconvolution of the experimental image. The TFI for one probe molecule is calculated and normalized to that for a restored image (CY3, 16250 ± 975; FITC, 5150 ± 600; and CY5, 14075± 1050). For exhaustive photon reassignment (EPR), the sampled PSF defines the depth (number of optical sections) over which light is integrated by the deconvolution process.
16
Fluorescent probes diluted in mounting media (2 pg/μl) were allowed to adsorb to the surface of a cover slip and then optically sectioned. The restored images revealed the distribution of intensities attributed to individual probes. The empirical measurements of the TFI for one probe either in solution or immobilized on glass cover slips were compared with a theoretical estimate of the number of photons emitted by one dye molecule by using the molar extinction coefficient, the quantum yield, the measured light flux of the microscope, and the quantum efficiency of the CCD camera. This estimate came within 80% of the actual measurement obtained.
17
For cell culture and in situ hybridization, NRK cells (djf clone from parent cell line, NRK-52E, American Type Culture Collection CRL 1571) were grown to confluence on 22 mm by 22 mm acid-washed cover slips in F12K media supplemented with 10% fetal bovine serum (FBS). The djf clone showed serum-responsive β- and γ-actin genes. For serum induction experiments, cells were incubated with F12K media (0.5% FBS) for 24 hours and then stimulated with 10% FBS. Cells were fixed for 10 min at room temperature in 4% paraformaldehyde in phosphate-buffered saline (PBS) (2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4) after serum stimulation, washed, and stored in PBS. The FISH protocol was modified from K. L. Taneja and R. H. Singer [J. Cell. Biochem. 44, 241 (1990)]. Cells were hybridized for 3.5 hours at 37°C. Cover slips were washed and mounted on slides with phenylenediamine in 90% glycerol with PBS. Multicolored (FITC, tetramethyl rhodamine isothiocyanate), 0.099-μm-diameter latex beads (Molecular Probes) were included in the mounting media and used as fiduciary markers to align images.
18
To elucidate the true fluorescence distribution of the acquired image, we restored the series of optical sections by using an iterative algorithm (Exhaustive Photon Reassignment, EPR) that used non-negativity constraints. Images were restored with 400 iterations and a smoothing factor alpha value = 5 × 10−6and a convergence value of 0.001. The procedure has been detailed previously [W. A. Carrington, et al., Science 268, 1483 (1995); Taneja K. L., Lifshitz L. M., Fay F. S., Singer R. H., J. Cell Biol. 119, 1245 (1992)].
19
Background in a restored image comprises point sources of low-level autofluorescence, light scattering, and random noise contributed by the imaging electronics. A threshold value was established and applied to the restored images to eliminate these low-level point sources of light by analyzing the background TFI frequency distribution of nonzero pixels in a cell subjected to a mock hybridization. The mean TFI per nonzero voxel +3 SD was chosen as the threshold value; all pixels less than the threshold value were set to zero, whereas those greater were retained at their original value. This eliminated >95% of the low-TFI background pixels. The threshold value was <10% of that predicted for one probe molecule (five CY3 fluorochromes) and when applied to the experimental image did not adversely affect the value from one hybridized probe. Brighter background pixels that eluded the thresholding filter but whose TFI value was less than that for one hybridized probe were excluded during the mapping analysis. The result was a filtered image where one hybridized CY3 labeled probe could be distinguished.
20
Data Analysis and Visualization Environment (DAVE); copyright 1995 by Lawrence M. Lifshitz and the University of Massachusetts Medical School.
21
The reassignment of light to a point source in a restoration results in one or two brighter voxels at the location of the point source in addition to a number of associated contiguous voxels. A point source is thus described as an object comprising a group of contiguous nonzero voxels in a restored image after thresholding. All the nuclear and cytoplasmic point sources therefore had a finite size and were treated as three-dimensional objects.
22
Additional evidence that every discrete signal is a single RNA molecule is that, when each of the five probes was hybridized individually, analysis showed that >95% of the detected sites had one probe hybridized. When two probes to the 3′-UTR were used, a population of single and double probes was detected (6). As an additional control, a set of five probes to an mRNA not expressed in these cells (α-actin) gave about 20 single probe signals per cell (2% of the β-actin single probe signal), but no multiple probe signals when processed by identical methods.
23
Supplementary material is available at http://www.sciencemag.org/feature/data/975399.shl . General information is available at http://www.singerlab.aecom.yu.edu.
24
We thank members of the Biomedical Imaging Facility, L. M. Lifshitz and J. Collins for their assistance with the DAVE software, R. A. Tuft for carrying out the light-flux measurements of the microscope, Y.-L. Wang for NRK cells, and K. Taneja for oligonucleotide probe synthesis. Supported by NIH grant GM 54887 to R.H.S. We are deeply saddened by the loss of our colleague and close friend Fredric S. Fay during the preparation of this manuscript.
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Science
Volume 280 | Issue 5363
24 April 1998
24 April 1998
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Received: 3 October 1997
Accepted: 20 February 1998
Published in print: 24 April 1998
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