Discontinuous transcription of ribosomal DNA in human cells.

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

Introduction

Numerous studies show that genes in all kinds of organisms, from prokaryotes to mammals, can be transcribed in short bursts or pulses alternated by periods of silence (reviewed in Smirnov et al. [1]) The probability of such mode of expression was suggested long ago;[2] now it seems that the discontinuous transcription is a common feature of the gene expression, at least in mammalian cells.[3-12] The periodical switches of the promoter between the active and “refractory” states may be crucial in the efficient regulation of the gene expression.[13-17] General considerations suggest even more significant role of the phenomenon in the dynamic organization of the cell, since the pulsing mode of one process is likely to be a cause and a consequence of pulsing in other processes. Thus, RNA processing, which is closely linked to the RNA synthesis, seems to be discontinuous.[9] A spontaneous heterogeneity of gene expression occasioned by transcriptional fluctuations may influence cell behaviour in changing environmental conditions and in the course of differentiation.[18]

The discontinuous character of transcription has been detected by various methods (reviewed in Smirnov et al. [1]) The number of transcripts produced in a certain (sufficiently short) period of time may be determined with high precision by single molecule RNA fluorescence in situ hybridisation (smFISH).[19-21] The results of such quantification alone provide indirect, but valuable information for modelling the expression kinetics in a cell population or tissue, when the studied gene is supposed to be transcriptionally active in all the cells. Methods based on the allele-sensitive single-cell RNA sequencing also allow to reveal and characterize the transcription bursting.[22] To monitor gene expression in real time, cells are transfected with constructs providing a fluorescent signal that corresponds to the expression of a particular gene. In a gene trap strategy, a luciferase gene is inserted under the control of endogenous regulatory sequences. Since both the luciferase protein and its mRNA are short-lived, the method allows to calculate the key parameters of the transcriptional kinetics. Probably the most popular in vivo method is based on the use of bacteriophages derived fluorescent coat proteins, such as MS2 or PP7, fused with GFP, which allows to visualize a bunch of the nascent RNA molecules accumulated around one gene.[4, 23, 24]

So far, the pulse-like transcription is well documented only in the genes transcribed by RNA polymerase II. It is not clear yet whether ribosomal DNA (rDNA) is also expressed discontinuously. In human cells, the clusters of multiple rDNA repeats, known as Nucleolus Organizer Regions (NORs), are situated on the short arms of the acrocentric chromosomes. Each repeat includes a gene coding for 18S, 5.8S and 28S RNAs of the ribosomal particles and an intergenic spacer.[25-30] In the interphase nucleus the rDNA provides the basis for the formation of nucleoli. The transcription by pol I and the first steps of rRNA processing take place in the special nucleolar units (FC/DFC) composed of fibrillar centers (FC) and dense fibrillar components (DFC).[31-42] The units correspond in light microscopy to the “beads” forming nucleolar necklaces,[43-46] and each unit is believed to accommodate a single transcriptionally active gene.[33, 39, 47, 48] The intensity of the rDNA transcription is usually very high throughout the entire interphase, especially at the S and G2 phases.[49] Now most of the methods used for the detection of the transcription fluctuation are hardly applicable to the ribosomal genes, since one cell usually contains hundreds of such genes. An alternative method was designed for direct measurements of rDNA transcription in the live cells by using the label-free confocal Raman microspectrometry.[50] This work revealed an undulatory character of the ribosomal RNA production in the whole nucleoli. In our earlier study on tumour-derived cells expressing a GFP-RPA43 (a subunit of pol I) fusion protein, we have observed specific fluctuations of the fluorescence signal in the individual FC/DFC units.[51] We also found high correlation of pol I and incorporated FU signals within the units. These data suggested that the ribosomal genes are transcribed in a pulse-like manner.

In the present work we used a different approach to the study of the discontinuous transcription of ribosomal genes in human cells. In our experiments with 5-fluorouridine (FU) and BrUTP, we found that the nucleolar transcription can be efficiently arrested at +4°C and quickly restored at normal conditions. Based on this finding, we synchronized the pol I activity in the cell population by cold block and release. Then using specially designed software we measured the intensity of transcription signal (incorporated FU) in the nucleoli and individual FC/DFC units at different periods after the release. This enabled us to detect transcription fluctuations of ribosomal genes in tumour derived as well as normal human cells and to reveal special properties of this fluctuation.

Methods

Ethics

The study followed the standards of the Ethics Committees of the General Teaching Hospital and the First Faculty of Medicine of Charles University, Prague, Czech Republic (Ethics Committee of General Univeristy Hospital, Prague approval no. 1570/11 S-IV (held on October 13, 2011, and updated January 18, 2018. The name of project: Pathogenesis of hereditary, degenerative and systemic diseases with manifestations in the eye, transplantology. Study of healthy and control tissue), and adhered to the principles set out in the Helsinki Declaration. We obtained human cadaver corneoscleral rims from 10 donors, which were surplus from surgery and stored in Eusol-C (Alchimia, Padova, Italy), from the Department of Ophthalmology, General University Hospital in Prague, Czech Republic, for the study. On the use of the corneoscleral rims, based on Czech legislation on specific health services (Law Act No. 372/2011 Coll.), informed consent is not required if the presented data are anonymous in the form."

Cell cultures

Human limbal epithelial cells (LECs) were obtained from XY cadaver corneoscleral rims after cornea grafting at University Hospital Kralovske Vinohrady, Prague, Czech Republic. The mean donor age ± standard deviation (SD) was 63.5 ± 6.5 years. Tissue was stored in Eusol-C (Alchimia, srl., Ponte San Nicolò, Italy) preservation medium at +4°C. The mean storage time ± SD (from tissue collection until explantation) was 7.2 ± 3.6 days. The corneoscleral rims were prepared as described before.[52, 53] Shortly, corneoscleral rims were cut into 12 pieces and placed in a 24-well plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland) on Thermanox plastic coverslips (Nunc, Thermo Fisher Scientific, Rochester, NY, USA). Explants were cultured in 1 ml of complete medium [1:1 DMEM/F12, 10% FBS, 1% AA, 10 ng/ml recombinant EGF, 0.5% insulin-transferrin-selenium (Thermo Fisher Scientific), 5 μg/ml hydrocortisone, 10 μg/ml adenine hydrochloride and 10 ng/ml cholera toxin (Sigma-Aldrich, Darmstadt, Germany)]. The culture media were changed every 2–3 days until the cells were 90–100% confluent (after 2–4 weeks).

HeLa cells were cultivated at 37°C in Dulbecco modified Eagle's medium (DMEM, Sigma) containing 10% fetal calf serum, 1% glutamine, 0.1% gentamicin, and 0.85g/l NaHCO3 in standard incubators. For the transcription synchronization, the cells were incubated in cold medium (+4°C) for 1 h, then transferred to the normal conditions and fixed at different time points from 15 to 150 min with the interval of 15 min.

Plasmids and transfection

The GFP-RPA43 and GFP-Fibrillarin vectors were received from Laboratory of Receptor Biology and Gene Expression Bethesda, MD.[54] The constructs were transfected into HeLa cells using Fugene (Qiagen).

Labeling of the transcription sites

For visualization of the transcription sites, sub-confluent cells were incubated for 5 min prior to fixation with 5-fluorouridine (FU) (Sigma). The cells were fixed in pure methanol at -20°C for 30 min and processed for FU immunocytochemistry. BrUTP (Sigma) was introduced into cells by the scratch procedure.[55, 56] Here we followed the same procedure as for the labelling of replication in the cited works. Briefly, the cells were grown on the coverslips; a drop of medium containing 20μg/ml BrUTP was applied upon each coverslip; then the latter was scratched by the tip of a syringe needle and incubated for 5 min at 37°C. Thus permeabilized, the cells were incubated for 10 min in the usual medium and then fixed and processed as after the incorporation of FU.

Incorporated FU and BrUTP signal was visualized using a mouse monoclonal anti-BrdU antibody (Sigma) and secondary goat Cy3-conjugated anti-mouse antibody (Abcam).

Light microscopy

Confocal images were acquired by means of SP5 (Leica) confocal laser scanning microscope equipped with a 63×/1.4NA oil immersion objective. For in vivo cell imaging we used a spinning disk confocal system based on Olympus IX81 microscope equipped with Olympus UPlanSApo 100×/1.4NA oil immersion objective, CSU-X spinning disk module (Yokogawa) and Ixon Ultra EMCCD camera (Andor). The live cells were maintained in glass bottom Petri dishes (MatTek) at 37°C and 5% CO2 within a microscope incubator (Okolab).

Software and data analysis

For measurement and counting of the transcription and other signals corresponding to individual FC/DFC units in 3D confocal images, we developed a MatLab based software.[51] The program identifies each unit by creating a maximum intensity projection of the confocal stack and blurring the projection with a Gaussian filter (σ = 8–10 pixels), defining the blurred image with a value obtained by Otsu's method for automatic threshold selection. After that, the optical section whereupon the unit had maximum intensity was identified. The final result contains 3D coordinates of each unit, its size (full-width half-maximum), the value of χ2, and integral intensities in the spheres with radii 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 pixels. The values corresponding to 1.5 pixels seemed to be the most resistant to noise and were used for presentation of the data. FC/DFC units were counted after deconvolution with Huygens software.

For measuring signals in the entire nucleoli we used a custom ImageJ plugin available at https://github.com/vmodrosedem/segmentation-correlation.[45] Based on the confocal stacks, the program identifies the regions occupied by the cell nuclei as well as nucleoli, measures their areas (in pixels), and the intensities, both integral and average, of the signal within these areas.

Results

1. Effects of low temperature on the nucleolar transcription

In the control the incorporated FU is accumulated predominantly in the nucleolar beads which, according to our earlier study,[55] correspond to the FC/DFC units of the nucleoli (Fig 1). The transcription signal in the nucleoplasm appeared as multiple small foci of much lower intensity. After 15 min of incubation at +4°C (without additional supply of CO2), both HeLa and LECs lost the ability to incorporate 5-fluorouridin (FU). When the cells were returned to the normal conditions (37°C, 5% CO2), transcription was partly restored in 15 min, and in 30 min the FU incorporation did not visibly differ from the control (Fig 1).

Fig 1

(A) Transcription in HeLa cells is quickly inhibited at +4°C and restored at the normal conditions. The transcription signal (FU incorporation) is accumulated in the nucleoli. The signal disappeared after 15 min of cold treatment (top right); when the cells were transferred to the normal conditions, the signal was partly restored in 15 min and appeared like in the control in 30 min (bottom). (B) Incorporation of BrUTP. No significant signal in the cells fixed after the permeabilization immediately (left) or following 10 min of incubation at +4°C (middle); when permeabilization was followed by 10 min incubation at +37°C, specifically labelled cells could be observed along the scratch track (right). Scale bar: 10μm.

Since incorporation of FU is preceded by its penetration in the cell and phosphorylation, we performed an additional experiment with another RNA predecessor, BrUTP, which was introduced in the HeLa cells by the scratch procedure (Fig 1B).[55, 56] When cells were permeabilized by scratching in the presence of BrUTP for 5 min and then immediately fixed (Fig 1B, left) or washed and incubated for further 10 min at +4°C (Fig 1B, middle), there was no significant incorporation of the nucleotide. But when the permeabilization was followed by 10 min incubation in the normal conditions (Fig 1B, right), the cells situated along the scratch track displayed the transcription signal in the nucleoli and nucleoplasm. This result confirmed that the transcription was efficiently arrested in our experiments at +4°C.

It is known that pol I and fibrillarin are particularly sensitive to stress, and their redistribution in the cell nuclei is a common symptom of nucleolar pathology. Therefore, to assess the effect of cold on the FC/DFC units, which are the centers of rDNA transcription and early rRNA processing, we transfected the cells with GFP-RPA43 or GFP-Fibrillarin. At the low temperature the GFP-Fibrillarin signal did not change significantly, but the intensity of the RPA43 signal was decreased as average to about 60% of the control level (Fig 2).

Fig 2

Following GFP-RPA43 and GFP-Fibrillarin signals in the transfected HeLa cells in vivo.

The intensity of the GFP-RPA43 signal is reduced after 15min incubation at +4°C (left, top) and restored after subsequent 30 min incubation at normal conditions (top, right). The GFP-Fibrillarin signal was not significantly affected by the cooling/warming procedure (bottom). Scale bar: 5μm.

Observation of the individual cells also showed that after transferring the cells from the cold to the normal conditions, the intensity of GFP-RPA43 signal in all FC/DFC units increased, although the number of the detectable units did not change (Fig 3).

Fig 3

Effects of cooling/warming (as in Fig 2, top) on the FC/DFC units in vivo in the transfected HeLa cells.

(A) intensity of the GFP-RPA43 signal in the individual units after 15min incubation at +4°C (black columns) and after subsequent 30 min incubation at 37°C (grey columns). Five cells were observed, and five selected units were followed in each cell. The error bars show SEM. (B) the total number of the GFP-RPA43 positive units in five cells after 15min incubation at +4°C (black columns) and after subsequent 30 min incubation at 37°C (grey columns). The experiment indicates that at the low temperature pol I escapes from the FC/DFC units.

These experiments show that low temperature causes a quick inhibition of the rDNA transcription, as well as significant though not complete depletion of the pol I pools in the nucleoli.

On the other hand, we observe a quick recovery of the cells without any lasting symptoms of pathology.

2. Synchronization of the nucleolar transcription in HeLa and human limbal cells by cold treatment

The experiments described in the previous section indicate that at the low temperature the ribosomal genes are brought to a silent state with a diminished RPA-GFP signal within the FC/DFC units which implies a decreased number of pol I complexes bound to the genes. This synchronization procedure was used for the study of the discontinuous expression of the rDNA in HeLa and LEC cells. Namely, the cells were incubated in cold medium (+4°C) for 1 h, then transferred to the normal conditions and fixed at different time points from 15 to 150 min with the interval of 15 min. FU was added to the cultivation medium 5 min prior to each fixation. The transcription signal visualized by antibody was then measured in the nucleoli by means of the ImageJ plugin software (see Methods). The results are presented in Fig 4.

Fig 4

Fluctuation of the intensity of the transcription signal (incorporated FU) in the whole nucleoli and nucleoplasm of HeLa cells after release from the cold block.

(A) Data of the individual experiments and the box-plot chart. (B) Mean values of the transcription signal intensity in the nucleoli after release from the cold block (left, top) and in the nucleoli of the control cells, i.e. without cold treatment (right, top) In the experiment the signal reaches maximal values at 30 min, 90 min, and 150 min. The graph shows mean values obtained from 50 cells in one experiment. Such experiment was repeated 8 times. CV- coefficient of variation. The error bars show SEM. The bottom graphs show the respective periodograms for the experiment (left) and control (right) calculated as amplitudes of the Fourier transforms. The x-axis represents the period (min). (C) Mean values of the transcription signal intensity in the nucleoplasm after release from the cold block (top) and the respective periodogram (bottom).

In all such experiments the intensity of the transcription signal increased during the first 30 min, then began to decrease. Altogether two cycles of rise and fall have been observed within the period of 150 min, the coefficient of variation (CV) was 0.26. The spectral analysis revealed a significant peak corresponding to the period of 60 min. Since the interval between the measurements was 15 min, the values of the period may be varying from 45 min to 75 min. An additional lower peak at 15 min probably reflected a high frequency noise. In the control, when the cells were kept at 37°C and fixed at different time points as in the experiment, the fluctuations of the transcription signal intensity were irregular. CV was only 0.07, and the periodogram had two peaks of low amplitude (compare the left and right parts of the Fig 4). In two experiments the period of observation was extended to 210 min, but between 150 and 210 min the fluctuations of the transcription signal appeared irregular with the CV values 0.06, i.e. just like in the control, which indicated that the synchrony in the cell population was lost. These results showed that in HeLa cells the activity of pol I transcription machinery was synchronized by the cold treatment for the period of 150 min, but not longer.

The same experimental procedure was applied to the LECs (Fig 5). In this case the first two cycles were more pronounced and the difference between control and experiment was more significant (compare Fig 5 and Fig 4). Otherwise, the dynamics of the transcription activity after the cold treatment proved to be similar in the studied cell lines. In the LECs, the periodogram had a more distinct peak at 60 min, but the synchronization also did not last longer than 150 min. CV was 0.29, i.e. slightly higher than in HeLa cells. It seems worth mentioning that our attempt to synchronize the transcription in human fibroblasts failed, for only a few of these cells recovered quickly enough after the cold treatment.

Fig 5

Fluctuation of the intensity of the transcription signal (incorporated FU) in the whole nucleoli and nucleoplasm of the limbal cells.

(A) Data of the individual experiments and the box-plot chart as in Fig 4A. (B) Mean values of the transcription signal intensity in the nucleoli after release from the cold block (left, top) and in the nucleoli of the control cells (right, top). The figure is analogous to the Fig 4. But in this case, the undulating pattern in the experiment (top, right) is more pronounced, and the periodogram related to the experiment (bottom, left) has a more distinct peak at 60 min. The data are obtained from 8 independent experiments, and in each of them 50 cells were measured. (C) Mean values of the transcription signal intensity in the nucleoplasm after release from the cold block (top) and the respective periodogram (bottom).

Thus, our experiments indicated that transcription of the ribosomal genes proceeds in a wave-like manner, although the employed synchronization procedure is not equally efficient in various cells.

3. Fluctuation of the pol I signal in the cells synchronized by chilling

To confirm our result by an independent set of data, we used chilling shock to synchronize HeLa cells transfected with GFP-RPA-43 (see Fig 6). Measuring the intensity of the pol I signal in the nucleoli of the individual cells, we observed fluctuations similar to those of the transcription signal (Figs 4 and 5). The fluctuations had a relatively low amplitude, and the distinct undulations persisted for no longer than two hours, apparently because only a minor portion of the pol I molecules within FC/DFC units are engaged in the current transcription, as was indicated, for instance, in our earlier work.[51] Nevertheless, the initial increase of the signal intensity during the first 30 min after the cold treatment was followed by a noticeable decrease during the next half hour, which could not be attributed to the effects of recovery. Together with the other results of the present study (Figs 4 and 5) this indicates, that fluctuations of the transcription intensity and pol I levels in the nucleoli are synchronous.

Fig 6

Fluctuation of the intensity of GFP-RPA43 signal in the nucleoli of HeLa cells after the cold treatment.

The eight successive images of the same transfected cell photographed every 15 min after the release from the cold block. The intensity of the signal at 15 min as well as at 60 min after the release is visibly lower than at other points. The graph at the bottom right shows records of the pol I signal intensity in five cells at different time points after the release. Each curve represents one cell. All curves have two peaks at 30 min and at 90 min or close to it, as in the case of FU incorporation (Figs 4 and 5). Scale bar: 5μm.

4. Synchronization of the transcription in the nucleoplasm by cold treatment

When the LECs or HeLa cells were incubated at +4°C, the transcription ceased completely in their nucleoplasm as well as in the nucleoli. Measurement of the total FU signal after transferring the cells from the cold to the normal conditions showed symptoms of synchronization: the signal in the nucleoplasm increased for 30 min and then began to decrease (Figs 4A, 4C, 5A and 5C). The average intensity of the transcription signal in the nucleoli and nucleoplasm positively correlated, with the correlation coefficients 0.65 for the HeLa cells and 0.74 for the LECs. But, as one could expect, the total expression of the nucleoplasmic genes was less synchronized. After the initial recovery and subsequent decrease, the signal became rather noisy. The CV was 0.17 and 0.19 in the HeLa and LECs respectively. The periodograms showed a not very distinct peak at 75 min as well as a sharper peak corresponding to higher frequencies. The second peak probably reflects a noisier character of the fluctuations in the nucleoplasm as compared to the nucleoli.

5. The FC/DFC units in the course of the transcription fluctuation

Since the measurement of the transcription signal in the whole nucleoli is significantly affected by the fluorescence between the FC/DFC units, we measured the signal also within the units. According to the data presented in the sections 2 and 3 (Figs 4 and 5), the intensity of FU signal in the nucleoli at 15 min and 30 min after the cold treatment may be taken as representatives of the two extreme states of the transcriptional fluctuation in the synchronized cells. Measurement of the FU signal in the individual FC/DFC units of the LECs and HeLa cells using the MatLab based software (see Methods) showed an approximately threefold increase of the signal intensity between 15 min and 30 min (Fig 7). But the transcription signal never disappeared from the cells completely, so that the average number of the FU-positive FC/DFC units did not change significantly (Fig 7B, right chart).

Fig 7

The transcription signal (incorporated FU) in the FC/DFC units of the limbal epithelial cells after the cold treatment.

(A) no FU incorporation in the cells incubated at +4°C for 15 min (left); a weak FU signal in the cell incubated at 37°C for 15 min after the chilling (middle); 30 min; completely recovered FU signal in the cell incubated at 37°C for 30 min after the chilling (right). Scale bar: 5 μm. (B) the average (from 50 cells) intensity of the FU signal in the individual FC/DFC units measured in the cells incubated at 37°C for 15 min and 30 min after the chilling. The increase is statistically significant (P < 0.0001, according to the Student’s t-test) (left). The average (from 50 cells) number of the FU positive FC/DFC units in the cells incubated for 15 min and 30 min at +37°C after the cold treatment; the differences are statistically insignificant (right). The data show an initial quick recovery of the transcription in the units without changing their number (see Fig 5).

Discussion

In our experiments, when the human derived cells were incubated at +4°C, transcription in their nuclei seemed to be arrested completely (Figs 1 and 7). At the same time the pol I signal in the FC/DFC units of the nucleoli was significantly reduced (Figs 2 and 6), whereas the amount of fibrillarin, which is an essential component of the early rRNA processing, did not change significantly (Fig 2). On the other hand, previous studies, including our own, indicate that the mobile fraction of pol I, apparently responsible for the actual transcription, constitutes less than a half of the entire pool of the enzyme in the units.[51, 54] Therefore, in all probability, the pol I complexes do not “freeze” on their matrices after the arrest of the transcription by the chill shock, but rather detach themselves and escape from the units. After returning to normal conditions, the pools of the enzyme are swiftly restored, and the rRNA synthesis in the cells is synchronized. This effect was used in our work for detection of the pulse-like transcription.

In our previous work we studied the fluctuations of pol I signal, but could not speak about the discontinuous transcription otherwise than hypothetically, since the dynamics of this signal does not necessarily reflect the transcription.[55] Therefore, only after developing the cold/release method of cell synchronization, we obtained the data related to the transcription fluctuations directly.

In thus synchronized HeLa and LEC cells, we observed a wave-like modification of the nucleolar transcription signal with two successive peaks (Figs 4 and 5). It should be mentioned, that the recovery process, which seemed to be limited to the first 30 min after the cold treatment, could not account for the observed dynamics, especially the regularly observed decrease of transcription intensity after the initial increase, as well as more or less distinct second peak. In both kinds of cells, the predominant fluctuation period estimated by the spectral analysis was about 60 min. A similar value of the period was obtained in our previous work for the fluctuations of the GFP-RPA43 signal.[51]. After the two distinct cycles, the waves were damped; probably because of their irregularity and variability in the individual cells. Nevertheless, our data indicate that the ribosomal genes are expressed discontinuously, with intervals of 45–75 min between the bursts.

In our review on the discontinuous transcription, we indicated what seemed to be four main patterns in which this phenomenon may be manifested: the typical busts; the undulating pattern; the regular pulsing; and the rare transcription events.[1] As mentioned above, the fluctuations observed in our study do not seem to belong to the regular type. Rare events also must be excluded, since rDNA transcription is very intensive throughout the entire interphase. The typical bursts are separated by the relatively long periods of silence. But we observed no diminishing of the number of FU positive (Fig 7B) or pol I positive (Fig 3) FC/DFC units in the course of the experiment, although the mean intensity of the incorporated FU signal in the individual units was greatly reduced at the points of minimal transcription activity (Fig 7B). Therefore, the observed fluctuation of rDNA transcription most likely belongs to the undulating pattern, in which the bursts are alternated by periods of relatively rare transcription events.

Additionally, our method of synchronization allowed us to obtain averaged data concerning the fluctuations in the nucleoplasmic genes, since their expression was also inhibited by the cold treatment. After this procedure, the total transcription signal in the nucleoplasm showed symptoms of fluctuations with two discernible, though not very distinct, peaks (Figs 4A, 4C, 5A and 5C). Evaluating these results, we have to keep in mind that various nucleoplasmic genes in the same cell display a wide range of transcriptional kinetic behavior (reviewed in Smirnov et al. [1]).[4, 10, 57, 58] Moreover, some of these genes are expressed in typical bursts with long periods of silence, during which they cannot be detected by FU incorporation. We should also mention that the status of the nucleoplasmic RNA polymerases at the low temperature was not examined in our experiments, and thus we do not know how efficiently the transcription was synchronized. Nevertheless, the presence of two significant peaks on the periodograms (Figs 4C and 5C) suggests that numerous genes in the nucleoplasm were transcribed in a pulse-like manner with periods close to 15 min and 75 min.

Thus, our results indicate that ribosomal genes in human cells are expressed discontinuously, and their transcription follows undulating pattern with predominant period of about 60 min.

24 Oct 2019

PONE-D-19-25095

Discontinuous transcription of ribosomal DNA in human cells

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper by Eugene Smirnov et al. describes the analysis of rDNA expression during about 2 h after release of human cells from cold treatment. FU incorporation was reduced by the cold treatment and the signal reached the normal level after 30 min of incubation at 37°C.

Using transfected plasmid carrying Pol I gene fused with GFP or GFP-Fibrillarin construct they observed that cold treatment reduced the levels of Pol I in the FC/DFC units. In subsequent experiments they demonstrated that there are fluctuations of incorporated FU in the whole

nucleoli during 120-150 min.

The experimental data confirm the conclusion that rDNA genes are transcribed discontinuously during short period after incubation at 37°C.

The paper should be re-written. Authors should first describe all experiments in the text in more detail. E.g., why they use this particular experiment in the text. why they use the transfections with Pol I gene and Fibrillarin gene. How they measure the intensity and amplitude. What are a.u. and a.e. units? What secondary Ab were used in FU detection? Each panel in the Figure should be described.

Reviewer #2: The work describes the study of discontinuous expression of rRNA genes. This manuscript builds on their previous work published in the Nucleus journal. In a new work, the authors investigated discontinuous expression of ribosomal genes after inhibition of transcription using cold stress. It seems that the data presented are not enough to convincingly illustrate the assumptions made.

1. The main emphasis in the work is made on the analysis of the intensity of label (FU) incorporation into the nucleoli of cultured cells. However, the inclusion of FU depends not only on the intensity of expression, but also on the rate of its cellular uptake and phosphorylation in the cell. Therefore, confirmation of this key result by an independent method is necessary. The authors show in Fig. 2 and 3A (and in their previous work, this was also demonstrated) that the nucleolur accumulation of RPA 43 also changes with time. It is possible to confirm the presence of waves of intensity of ribosomal RNA expression using this method. The live cell imaging of RPA 43 during during the recovery of cells after cold stress (with estimations and statistical analysis) can illustrate described waves more accurately than the estimation of FU incorporation for overall cell population.

2. The authors indicate that the data on fluctuations and transcription cycles were obtained by averaging over several experiments (8 repetitions). It seems that it is necessary to present not only the result of averaging over 8 experiments, but also the curves for all individual experiments (plus, the result of averaging). To appreciate how is the distribution of averaged values among the time points, instead of using bar graphs the data should be represented using box-plots with the individual values as dots. Also, the statistical analysis of detected fluorescence intensity fluctuations should be presented. These changes were statistically significant or not?

3. I think that the experimental model used (restoration of transcription after cold stress) cannot be interpreted as synchronization. For human cells, this is very severe stress. And it seems that the subsequent processes should be interpreted as a process of recovery from stress. In this case, the transcription fluctuations can be connected with the process of cell restoration, which may differ from fluctuations in the control culture (which was described in the article in the Nucleus journal). Authors should at least briefly discuss such an interpretation of their data.

Reviewer #3: The main goal of the study by Smirnov et al. “Discontinuous transcription of ribosomal DNA in human cells” was to evaluate nucleolar and nucleoplasmic transcription in two types of human cells (HeLa and epithelial limbal cells) after their synchronization with a low (4o C) temperature followed be the release from the cold shock as compared with untreated controls. Aims were reached by incubation of cells with 5-fluorouridine as precursor of RNA synthesis, expression of plasmids encoding RNA pol I subunit (RPA43) and fibrillarin fused with GFP. The intensity and number of signals were examined using a MatLab based software [ref 51] and Image J facilities. The authors concluded that: (1) chilling of cells results in arrest of pol I (nucleolar) and pol II (nucleoplasmic) transcription but does not displace all pol I complexes from their intrinsic locations; (2) cell release from the chilling conditions restores rDNA transcription to control values; (3) the restoration of rDNA transcription follows a wave-like manner (within 15-210 min of observation) and is discontinuous process.

Comments: The major question concerns the principal novelty of the reviewed paper as compared with the papers recently published by the same authors in “Nucleus” (M. Hornáček et al., Fluctuations of pol I and fibrillarin contents of the nucleoli. Nucleus, 2017, 8: 421-432; Smirnov et al., Discontinuous transcription Nucleus, 2018, 9: 149-160). In both publications, it is stated that ribosomal genes, like other genes, are transcribed in pulse-like manner (e.g., Hornáček et al., 2018, page 150), while it is well known that rRNA genes are transcribed during the entire cell cycle [ref. 49], which duration is much longer (roughly 24 hours) than the duration of observations in the current study (150-210 min).

Minor questions:

Lines 25, 89 “in the populations of tumour derived” – Unclear meaning

Line 92: Methods

Conditions for cell chilling should be described in this section instead of Results.

Lines 159, 160 (Fig 1) – It is unclear, where are the nuclear boundaries, and how the authors determined that “… FU is accumulated predominantly in the FC/DFC units of the nucleoli” without using any markers for FC/DFC?

Lines 187, 190 (legend for Figure 3): “bars” should apparently be replaced by “columns”.

Fig.3A: What are the vertical bars: SEM or standard deviation (Ϭ).

Fig. 3B: SEMs (or Ϭ) are not indicated.

Lines 193, 194: It is remained unspecified how the authors distinguish between negative labeling of cells with 5-FU (5-fluorouridine) caused by inhibition of rDNA transcription from non-penetration of the precursor in cells in cold conditions. In addition, before incorporation in nascent pre-rRNA FU must be bound to ATP and this process most likely is suppressed by a low temperature. By other words, 5-FU was unincorporated not because genes were not transcribed, but because the precursor was inaccessible to nascent RNAs upon cold conditions.

Fig. 4 (Lines 207-213).

A – there are no SEM (or Ϭ) on the columns and therefore it is impossible to compare differences between various time-points statistically. The latter makes the authors statement about a fluctuating manner of rDNA transcription uncertain.

B – on the periodograms, the horizontal axis scale does not correspond to the relative graphs in Fig. 4A.

Fig. 5 (Lines 241-246): See comments to Fig. 4.

Fig. 6 (Lines 264-267): See comments to Fig. 4.

The images below Fig. 7 are not described (are they copies?).

Unfortunately, figures are not numbered that makes their identification complicated.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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6 Dec 2019

Our specific replies to the reviewers are as follows (here the remarks requiring our reply are put in bold font):

Reviewer #1: The paper by Eugene Smirnov et al. describes the analysis of rDNA expression during about 2 h after release of human cells from cold treatment. FU incorporation was reduced by the cold treatment and the signal reached the normal level after 30 min of incubation at 37°C. Using transfected plasmid carrying Pol I gene fused with GFP or GFP-Fibrillarin construct they observed that cold treatment reduced the levels of Pol I in the FC/DFC units. In subsequent experiments they demonstrated that there are fluctuations of incorporated FU in the whole nucleoli during 120-150 min.The experimental data confirm the conclusion that rDNA genes are transcribed discontinuously during short period after incubation at 37°C.

The paper should be re-written. Authors should first describe all experiments in the text in more detail. E.g., why they use this particular experiment in the text, why they use the transfections with Pol I gene and Fibrillarin gene.

In the revised version of the text we furnished our description with further details. In particular, we observed that, since redistribution of pol I and fibrillarin in the cell nuclei is a common symptom of nucleolar pathology, the study of cells transfected with GFP-RPA43 or GFP-Fibrillarin was needed to assess the reaction of the cells to the chilling (Results, 1st section, 3rd paragraph).

How they measure the intensity and amplitude. What are a.u. and a.e. units?

As in our previous works, we measured the signal intensity in arbitrary units (a.u.), but “a.e.“ in Fig 7 was an error, which we corrected in the revised text. “Amplitude” refers to the amplitude of the Fourier transform, i.e. the value of the periodogram. This was mentioned in the legend to the Fig 4 of our original manuscript.

What secondary Ab were used in FU detection?

We indicated the antibody in the revised Methods.

Each panel in the Figure should be described.

We extended and corrected the description of the Figures.

Reviewer #2: The work describes the study of discontinuous expression of rRNA genes. This manuscript builds on their previous work published in the Nucleus journal. In a new work, the authors investigated discontinuous expression of ribosomal genes after inhibition of transcription using cold stress. It seems that the data presented are not enough to convincingly illustrate the assumptions made.

1. The main emphasis in the work is made on the analysis of the intensity of label (FU) incorporation into the nucleoli of cultured cells. However, the inclusion of FU depends not only on the intensity of expression, but also on the rate of its cellular uptake and phosphorylation in the cell. Therefore, confirmation of this key result by an independent method is necessary. The authors show in Fig. 2 and 3A (and in their previous work, this was also demonstrated) that the nucleolar accumulation of RPA 43 also changes with time. It is possible to confirm the presence of waves of intensity of ribosomal RNA expression using this method. The live cell imaging of RPA 43 during during the recovery of cells after cold stress (with estimations and statistical analysis) can illustrate described waves more accurately than the estimation of FU incorporation for overall cell population.

We did the confirming experiments on the GFP-RPA43 transformed cells, as suggested by the reviewer. The results are described in the text (Results, section 4) and shown in the Fig 6 of the revised manuscript.

2. The authors indicate that the data on fluctuations and transcription cycles were obtained by averaging over several experiments (8 repetitions). It seems that it is necessary to present not only the result of averaging over 8 experiments, but also the curves for all individual experiments (plus, the result of averaging).

In the revised manuscript we show the curves corresponding to individual experiments as well as the result of the averaging with the errors (Fig 4B,C and 5B,C of the revised manuscript).

To appreciate how is the distribution of averaged values among the time points, instead of using bar graphs the data should be represented using box-plots with the individual values as dots.

We created the box-plots, as suggested by the reviewer (Fig 4A and 5A of the revised manuscript).

Also, the statistical analysis of detected fluorescence intensity fluctuations should be presented. These changes were statistically significant or not?

We added statistical data (significance levels according to the Student’s t-criterion) showing significant change in the intensity of the FU signal and insignificant difference in the number of FC/DFC units between 15 min and 30 min after the cold treatment (see legend to the Fig 7)

3. I think that the experimental model used (restoration of transcription after cold stress) cannot be interpreted as synchronization. For human cells, this is very severe stress. And it seems that the subsequent processes should be interpreted as a process of recovery from stress. In this case, the transcription fluctuations can be connected with the process of cell restoration, which may differ from fluctuations in the control culture (which was described in the article in the Nucleus journal). Authors should at least briefly discuss such an interpretation of their data.

We provide a brief discussion suggested by the reviewer (see Results, 1st section, last paragraph and Discussion, 2nd paragraph). There was indeed a recovery process, but it seemed to be limited to the first 30 min after the chilling, and it could not account for the regularly observed decrease of transcription intensity at 60 and 120 min (see Fig 4 and 5).

We would also observe that chilling does not seem to be a severe stress for the cultivated cells. Unlike the common methods of transcription inhibition (such as actinomycin, amanitin or DRB treatment), it causes no lasting abnormalities; in fact, we did not observe any changes in the nucleolar distribution of fibrillarin after the cold treatment (see Fig 2).

Relative mildness of the chilling shock may also be seen from the fact that cells are well preserved when kept outside the incubator (e.g. during transportation) at low temperature (without freezing).

Reviewer #3: The main goal of the study by Smirnov et al. “Discontinuous transcription of ribosomal DNA in human cells” was to evaluate nucleolar and nucleoplasmic transcription in two types of human cells (HeLa and epithelial limbal cells) after their synchronization with a low (4o C) temperature followed be the release from the cold shock as compared with untreated controls. Aims were reached by incubation of cells with 5-fluorouridine as precursor of RNA synthesis, expression of plasmids encoding RNA pol I subunit (RPA43) and fibrillarin fused with GFP. The intensity and number of signals were examined using a MatLab based software [ref 51] and Image J facilities. The authors concluded that: (1) chilling of cells results in arrest of pol I (nucleolar) and pol II (nucleoplasmic) transcription but does not displace all pol I complexes from their intrinsic locations; (2) cell release from the chilling conditions restores rDNA transcription to control values; (3) the restoration of rDNA transcription follows a wave-like manner (within 15-210 min of observation) and is discontinuous process.

Comments: The major question concerns the principal novelty of the reviewed paper as compared with the papers recently published by the same authors in “Nucleus” (M. Hornáček et al., Fluctuations of pol I and fibrillarin contents of the nucleoli. Nucleus, 2017, 8: 421-432; Smirnov et al., Discontinuous transcription Nucleus, 2018, 9: 149-160). In both publications, it is stated that ribosomal genes, like other genes, are transcribed in pulse-like manner (e.g., Hornáček et al., 2018, page 150), while it is well known that rRNA genes are transcribed during the entire cell cycle [ref. 49], which duration is much longer (roughly 24 hours) than the duration of observations in the current study (150-210 min).

In the quoted work (Hornáček et al., 2017) we studied the fluctuations of pol I signal, but could not speak about transcription otherwise than hypothetically, since, for instance, our FRAP experiments indicated that most of the pol I molecules within FC/DFC units were not engaged in the current transcription. Only after developing the cold/release method of cell synchronization, we obtained the data related to the transcription directly. Hence, the straightforward title of our new study.

In the revised text we emphasized the novelty of the present work (Discussion, 2nd paragraph).

Minor questions:

Lines 25, 89 “in the populations of tumour derived” – Unclear meaning

We changed “tumour derived“ to “HeLa“

Line 92: Methods

Conditions for cell chilling should be described in this section instead of Results.

We added this description to the Methods, but left it also in the Results for the convenience of the reader

Lines 159, 160 (Fig 1) – It is unclear, where are the nuclear boundaries,

In the revised manuscript, we showed the outlines of the nuclei on the indicated Figure.

and how the authors determined that “… FU is accumulated predominantly in the FC/DFC units of the nucleoli” without using any markers for FC/DFC?

In the revised manuscript we provided the reference (Results, 1st section, 1st paragraph) to our previous publication (Smirnov et al, 2016), where we demonstrated colocalization of FU and pol I fluorescent signals, as well as correspondence of the latter to electron microscopic images of the FC/DFC units.

Lines 187, 190 (legend for Figure 3): “bars” should apparently be replaced by “columns”.

We made the correction.

Fig.3A: What are the vertical bars: SEM or standard deviation (Ϭ).

The error bars signified SEM. We indicated this in the legends of the revised manuscript.

Fig. 3B: SEMs (or Ϭ) are not indicated.

As the legend says, the Fig 3B represents the total numbers of the FC/DFC units in the five cells (unlike Fig 3A, which represents the average intensities). Thus there are no statistical errors in this case.

Lines 193, 194: It is remained unspecified how the authors distinguish between negative labeling of cells with 5-FU (5-fluorouridine) caused by inhibition of rDNA transcription from non-penetration of the precursor in cells in cold conditions. In addition, before incorporation in nascent pre-rRNA FU must be bound to ATP and this process most likely is suppressed by a low temperature. By other words, 5-FU was unincorporated not because genes were not transcribed, but because the precursor was inaccessible to nascent RNAs upon cold conditions.

In the revised version of the manuscript, we present the data of a new experiment, in which BrUTP was used instead of FU (See Results, 1st section, Fig 1B and Methods for the experiment description). The results seemed to show convincingly that cold alone inhibited the transcription in our experiments.

Fig. 4 (Lines 207-213). A – there are no SEM (or Ϭ) on the columns and therefore it is impossible to compare differences between various time-points statistically. The latter makes the authors statement about a fluctuating manner of rDNA transcription uncertain.

Fig. 5 (Lines 241-246): See comments to Fig. 4.

Fig. 6 (Lines 264-267): See comments to Fig. 4.

Combining the suggestions of the reviewers 2 and 3, we provided the error bars, as well as the graphs corresponding to individual experiments and box-plots in the Figs 4,5.

B – on the periodograms, the horizontal axis scale does not correspond to the relative graphs in Fig. 4A.

Our error was in applying the same term “time period” to different variables. In the revised text, to distinguish between the real time of the experiment and the parameter of the periodogram, we changed the first to ”Time“ and the second to ”Period.“

The images below Fig. 7 are not described (are they copies?).

Indeed, the images appeared in the pdf version as the result of an error.

Unfortunately, figures are not numbered that makes their identification complicated.

We added the numbers.

Submitted filename: Reviewers.docx

Click here for additional data file.

7 Jan 2020

PONE-D-19-25095R1

Discontinuous transcription of ribosomal DNA in human cells

PLOS ONE

Dear Dr. Smirnov,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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-clarifying statistical analysis results as pointed by Rev#2

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-clarifying the methods section as suggested by Rev#3

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Synchronized HeLa and LEC cells demonstrated two successive peaks of rDNA transcription. These picks are now presented for individual cells and for averaging.

The text and Legends to Figures in the revised paper include the details that help a reader to understand the paper.

All my concerns were addressed.

Reviewer #2: Minor questions

1. Line 336. The usual p-value in t-test is 0.05.

2. The data in Fig. 4A and 5A. Is each curve based on a measurement of 50 cells? Maybe then it is worth presenting for each point the data not only on the mean, but also the standard deviation?

3. The new figures are not embedded in the logic of the article. In the current version, the last panel with box-plot in fig. 4a and fig. 4b (+ last panel with box-plot in 5a and fig. 5b) duplicate each other.

4. It is not clear why the error bars for the control cells are not provided (Fig. 4B and Fig. 5B, right panels)?

Reviewer #3: Comments to the revised version of the paper by Smirnov et al. “Discontinuous transcription of ribosomal DNA in human cells”

In general, the authors took into account my comments and adequately answered the questions.

However:

(1) The Abstract remains almost unchanged. For example, it does not include mentioning the results of BrUTP experiments, which were included to the revised paper (lines 176-183). According to my opinion, these experiments are crucially important for interpretation of the FU-labeling data. The same is also true for the last paragraph of Introduction, where the authors summarize the main paper results.

(2) Methods

It remains unclear, why the BrUTP labeling protocol is described only “briefly” (line 135). The publications cited by the authors for the scratch procedure used in the current study [55, 56] apply cell labeling with DNA replication but not with any transcription precursors.

What was the duration of cell fixation with methanol (line 133)?

What was the working concentration of BrUTP?

Line 140: “cy3-conjugated” should be replaced by “Cy3-conjugated”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

22 Jan 2020

Reviewer #1:

All my concerns were addressed.

Reviewer #2: Minor questions

1. Line 336. The usual p-value in t-test is 0.05.

We presented the result in the form suggested by the reviewer.

2. The data in Fig. 4A and 5A. Is each curve based on a measurement of 50 cells? Maybe then it is worth presenting for each point the data not only on the mean, but also the standard deviation?

We showed the deviations on the indicated graphs

3. The new figures are not embedded in the logic of the article. In the current version, the last panel with box-plot in fig. 4a and fig. 4b (+ last panel with box-plot in 5a and fig. 5b) duplicate each other.

The figure 4B contains mean values with standard deviations, whereas Fig 4A shows the extreme values of the set, the median and two other quartiles. We think that both indicated figures are useful.

4. It is not clear why the error bars for the control cells are not provided (Fig. 4B and Fig. 5B, right panels)?

This was only one experiment. It served to show that the variations of the transcription intensity in the control (the noise) were far exceeded by the variations in the experiment.

Reviewer #3:

(1) The Abstract remains almost unchanged. For example, it does not include mentioning the results of BrUTP experiments, which were included to the revised paper (lines 176-183). According to my opinion, these experiments are crucially important for interpretation of the FU-labeling data. The same is also true for the last paragraph of Introduction, where the authors summarize the main paper results.

We agree that these data are important, and we include them in the Abstract and Introduction of the second revised version.

.

(2) Methods

It remains unclear, why the BrUTP labeling protocol is described only “briefly” (line 135). The publications cited by the authors for the scratch procedure used in the current study [55, 56] apply cell labeling with DNA replication but not with any transcription precursors.

Applying the scratch method to the labelling of transcription we used BrUTP instead of DNA precursors. Otherwise, the procedure was the same as in the replication labelling. We clarify this point in the revised text.

What was the duration of cell fixation with methanol (line 133)?

The cells were fixed for 30 min. We provide this information in the revised text

What was the working concentration of BrUTP?

20µg/ml. We provide this information in the revised text.

Line 140: “cy3-conjugated” should be replaced by “Cy3-conjugated”.

We make this correction in the revised text.

Submitted filename: letter2.docx

Click here for additional data file.

27 Jan 2020

Discontinuous transcription of ribosomal DNA in human cells

PONE-D-19-25095R2

Dear Dr. Smirnov,

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

20 Feb 2020

PONE-D-19-25095R2

Discontinuous transcription of ribosomal DNA in human cells

Dear Dr. Smirnov:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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