A statistical framework for quantifying the nuclear export rate of influenza viral mRNAs

Figures

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  Figure 1.. Viral mRNA detection by single-colour RNA FISH. (A) A schematic diagram of mRNA labelling for the diffraction-limited imaging. Each mRNA was hybridised with a tile of oligonucleotides, each labelled with a single fluorophore at the 3′ end. (B) A representative field-of-view of the microscope image in which nucleoprotein (NP) mRNAs were stained with Quasar 670. White, single-molecule RNA FISH (smFISH) signal; blue, nucleus stained with DAPI. The red square indicates the area for the sequential close-up of the image. The signal intensity is shown on the same greyscale across the three panels, indicated on the right. (C) Spot detection in 3D. Spots are projected to either the XZ or YZ plane to indicate the distribution of spots in a typical 3D cell volume. The colour of each spot indicates the Z position of that spot. Spot projection to the XY plane at the bottom (magenta) highlights the XY positions of spots relative to the nucleus boundary (blue), i.e., nucleus projection on the XY plane. The grid interval on the X and Y axis is ca. 18.3 µm. (D) Total count of mRNAs in each cell. The sample size (n) indicates the number of cells segmented in the microscope images. (E)…

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  Figure 2.. Viral mRNA detection using multiplex RNA FISH. (A) An overview of the assay. The viral mRNAs were hybridised with the encoding probes, and then the amplification probes were annealed (not shown in the diagram for simplicity). A flow cell was assembled in which the readout probes were hybridised and the cells were imaged in the three sequential rounds of hybridisation and imaging. Subsequently fluorescent signals were decoded according to the encodings listed on the right. (B) A representative mapping of identified viral mRNAs on the segmented cell and nuclear boundary in the entire field-of-view (left) and the close-up in the red square (right). The mesh grids indicate ~34 µm interval on the left, and ~17 µm interval on the right. Cells were removed if their cytoplasmic boundaries touched any edge of the field-of-view. (C) Total count of mRNAs in 204 single cells (top row) and the nuclear fraction (bottom row) for each segment. (D) Heatmap representation of mRNA counts in single cells. The viral segments are in rows, cells in columns. The cells were clustered column-wise to highlight the inequality in the abundance of transcripts carried per cell. The colour code for the absolute…

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  Figure 2—figure supplement 1.. Independent validation of the total mRNA count and the nuclear ratio of each segment by single-molecule RNA FISH (smFISH). (A) A schematic diagram of in-house probe synthesis. The pool of single-stranded DNA templates were PCR-amplified, T7-transcribed, and reverse-transcribed using the fluorescent-labelled primer. The number of distinct probes contained in the pool is indicated for each segment. For HA, NA, M, and NS segments, two additional fluorescent probes bind to each probe. As a result, the number of fluorophores bound per mRNA molecule is theoretically three times as many as the probes for those segments. (B) Total count of mRNAs per cell for each segment. The sample size (n) indicates the number of cells analysed. (C) The nuclear ratio of mRNAs in each cell. The sample size is the same as the one indicated in panel B.

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  Figure 3.. Cell-to-cell heterogeneity for time-course inference. (A) A theoretical diagram of how viral mRNAs emerge in single cells in the population. The front runners designate ones in which the viral transcription was initiated earlier than others, and the slow starters are ones that started transcription lately. The bottom plot shows the theoretical relationship between the nuclear ratio and the abundance of transcripts in which the front runners and slow starters (and many more along the spectrum in between) compose the clusters as indicated. (B) Scatter plots of nuclear fraction against the total count in each cell, derived from the dataset presented in Figure 2C. (C) Time-course of the distribution of total nucleoprotein (NP) mRNA count per cell, measured by single-molecule RNA FISH (smFISH), at the indicated time points (top row); and the change in the nuclear fraction size, plotted against the total number of transcripts (bottom row). The sample size (n) indicates the number of cells observed at each time point.

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  Figure 3—figure supplement 1.. Independent validation of the nuclear ratio against the total count of mRNAs for each segment by single-molecule RNA FISH (smFISH). The nuclear ratio of mRNAs in each cell was plotted against the total number of mRNAs in that cell. The original data were derived from Figure 2—figure supplement 1B and C.

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  Figure 4.. Kinetic model for estimating the nuclear export rate. (A) A schematic diagram representing the production of viral transcripts in the nucleus and the export to the cytoplasm. The two spherical compartments are for the nucleus and the cytoplasm. Viral transcripts (magenta) are produced at a rate constant λ in the nucleus, and exported at a rate constant μ to the cytoplasm. (B) The relationship between the nuclear fraction and the total count in the kinetic model, according to the formula presented in panel A. The parameter α determines the curvature of the plot. (C) The parameter fit to the observed data points. The parameter α was estimated by fitting the equation to the observed data points presented in Figure 3B. The colour representation in each line is the same as the ones used in panel B, according to the parameter α. (D) Estimated nuclear export rate. The parameter μ was estimated from the three independent assays (indicated in black for each segment); the blue bar indicates the mean of the three measurements.

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  Figure 5.. Time-course experiment for viral mRNAs. (A) A schematic diagram of the time-course measurement. Cells were infected with the virus 2 hr prior to the baloxavir treatment for halting the viral transcription. Cells were fixed at 1-hr interval thereafter, then subjected to single-molecule RNA FISH. (B) The total mRNA counts over time post baloxavir treatment for the hemagglutinin (HA) (top) and nucleoprotein (NP) (bottom) segments. (C) The change of mRNA fraction detected in the nucleus over time post baloxavir treatment. The exponential decay was fitted against the data using either all the data points (indicated with the blue line) or the first two time points (indicated with the orange line). The exponents (i.e. the nuclear export rates) estimated are shown on the panel with the corresponding colour.

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  Figure 5—figure supplement 1.. Time-course measurement of viral mRNAs. The total mRNA counts over time post baloxavir treatment for the hemagglutinin (HA) and nucleoprotein (NP) segments. Two additional biological replicates accompanying the data presented in Figure 5B are shown.

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  Figure 5—figure supplement 2.. Viral mRNA export. The change of mRNA fraction detected in the nucleus over time post baloxavir treatment. The exponential decay was fitted against the data using either all the data points (indicated with the blue line) or the first two time points (indicated with the orange line). The exponents are shown inside the panel with the corresponding colour. These are two additional biological replicates accompanying the data in Figure 5C.

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  Figure 6.. Exclusive detection of unspliced NS transcripts. (A) Probe-binding sites on the NS segment. The exon probes, used in Figure 1, target the second exon of NS2 (spliced form), and the intron probes are against the region that is spliced out. Thus, exon probes detect both NS1 and NS2, while intron probes exclusively detect NS1. The black line represents the NS gene. The protein coding region is indicated with the wide horizontal bar. (B) The total spot count (left column) detected using either exon probes or intron probes. The spot nuclear ratio in each cell is indicated on the right. Note that the plots of the exon probes (orange) are the reproduction from Figure 1 and are presented for comparison with those of the intron probes (magenta).