Figure 2. Two distinct NDC80 transcripts are expressed during meiosis.

(A) Ribosome profiling and mRNA-seq reads over the NDC80 locus during vegetative growth (top track) or meiotic S phase (bottom track). Data are derived from (Brar et al., 2012). (B) NDC80 mRNA isoforms and Ndc80 levels in meiosis. NDC80^long and NDC80^short levels were determined by northern blot, and Ndc80 level was determined by anti-V5 immunoblot at the indicated time points. To induce meiotic entry, IME1 and IME4 expression was induced in the strain UB1337 by addition of CuSO₄ 2 hr after cells were transferred to SPO. SCR1, loading control for northern blot. Kar2, loading control for immunoblot. One of the two repeated experiments is shown. * indicates a smaller RNA product, which likely represents a truncated form of NDC80^long. (C) Representative smFISH images for NDC80^long and NDC80^short during vegetative growth and meiosis. Vegetative samples were taken when cells (UB8144) were growing exponentially in nutrient rich medium. Meiotic prophase samples were taken 6 hr after cells (UB8144) were transferred to SPO, a time when these cells were arrested in pachytene using the pGAL-NDT80 GAL4-ER system. Cells were then released by addition of β-estradiol, and meiosis I samples were taken 1.5 hr later. The Q 670 probes (shown in green) hybridize to the common region shared between NDC80^long and NDC80^short, whereas the CF 590 probes (shown in magenta) hybridize to the unique 5’ region of NDC80^long (schematic is shown in the right panel). DNA was stained with DAPI (blue). Each cell was staged by its Zip1-GFP signal. Vegetative growth: Zip1-GFP negative. Meiotic prophase: Zip1-GFP positive. Meiosis I: Zip1-GFP negative and post NDT80 induction. Images here and throughout are shown as the maximum-intensity projections of z-stacks. Scale bar: 5 µm. (D) Quantification of smFISH data shown in (C), graphed as the relative frequency histograms of cells with a given number of NDC80^long and NDC80^short transcripts per cell, using data pooled from three independent experiments. The dashed line indicates the median number of NDC80^long and NDC80^short transcripts per cell. Each histogram here and throughout was normalized so that the maximum bin height is the same across all histograms. A total number of 637 cells were analyzed for vegetative growth, 437 for meiotic prophase, and 491 for meiosis I. Two-tailed Wilcoxon Rank Sum test was performed between each pair of conditions as indicated by the bracket. Refer to Supplementary file 1F for a summary of the median transcript levels for all the smFISH experiments.

Figure 2—figure supplement 1. Ribosome profiling and mRNA-seq reads over the NDC80 locus, during vegetative growth, starvation (MATa/MATa), and throughout meiosis.

Data are derived from (Brar et al., 2012).

Figure 2—figure supplement 2. During starvation, NDC80^short and Ndc80 protein levels remain high, while NDC80^long is not expressed.

The abundance of NDC80 mRNA isoforms and Ndc80 protein level in starvation versus in meiosis. NDC80^long and NDC80^short were detected by northern blot, and Ndc80, by anti-V5 immunoblot. SCR1, loading control for northern blot. Pgk1, loading control for immunoblot. Cells harboring the pCUP-IME1 pCUP-IME4 system (UB1337) were transferred into SPO at 0 hr. After 2 hr, the culture was split into two: In one half, IME1 and IME4 expression was never induced, and thus cells stayed in starvation (A). In the other half, IME1 and IME4 expression was induced by addition of CuSO₄ (B). * indicates a smaller RNA product, which likely represents a truncated form of NDC80^long. Note: the samples in the induced conditions are the same as in Figure 2B, but the northern blot in this figure was a rerun of Figure 2B, and the immunoblot has a different loading control displayed.

Figure 2—figure supplement 3. Progression of cells through meiosis as determined by spindle morphology and DAPI staining.

(A) Percentage of cells with metaphase I, anaphase I, metaphase II, or anaphase II spindles at each time point of the experiment in Figure 2—figure supplement 2. (B) Percentage of cells with 1 (mononucleates), 2 (binucleates), or 3/4 (triad/tetranucleates) nuclei at the end of meiosis for the experiment in Figure 2—figure supplement 2, determined by counting cells stained with DAPI. In all analyses, 100 cells were counted per time point, per condition. Results from one of the two repeated experiments are shown.

Figure 2—figure supplement 4. Percentage of the colocalized or non-colocalized smFISH spots obtained using the odd and even smFISH probe sets.

Top: Schematic for the odd and even probe sets. Fifty-four oligonucleotide probes tiling the common region of NDC80^luti and NDC80^ORF were designed. All the odd number probes were labeled with one fluorophore (CAL Fluor Red 590, shown in magenta), and the even number probes, with another fluorophore (Quasar 670, shown in green). Wild type cells (UB8144) were transferred to SPO to induce meiosis. 6 hr later, samples were fixed and hybridized with the odd and even probe sets. A total of 428 meiotic prophase cells were analyzed, pooling from two independent experiments.

Figure 2—figure supplement 5. Bootstrapping analysis performed for the data obtained from the odd and even probe sets.

Quantified cells from all the acquired fields of view were pooled, and then a given number (n) of cells were randomly sampled 500 times. The mean and 95% credible interval were calculated for the fraction of paired and unpaired mRNA per cell, and these data plotted for each choice of the number n. The number of cells measured (428 cells) exceeded the number at which the error plateaued (60 cells), indicating that additional data would not improve the confidence in the measurements.

Figure 2—figure supplement 6. smFISH quantification for NDC80^long and NDC80^short in pre-meiotic starvation and meiotic prophase.

Cells harboring pCUP-IME1 pCUP-IME4 (UB6190) were transferred to sporulation medium (SPO), and after 2 hr in SPO, pre-meiotic samples were taken. CuSO₄ was then added to induce expression of IME1 and IME4. 2 hr after copper induction, meiotic prophase samples were taken. The quantification was graphed as the relative frequency histograms of cells with a given number of NDC80^long and NDC80^short transcripts per cell, using data pooled from three independent experiments. The dashed line indicates the median number of NDC80^long and NDC80^short transcripts per cell. A total number of 594 cells were analyzed for the pre-meiotic stage and 611 for meiotic prophase. Two-tailed Wilcoxon Rank Sum test was performed for NDC80^short and NDC80^long, respectively, comparing meiotic prophase with pre-meiotic starvation. Note: the frequency histogram for meiotic prophase is the same as in Figure 4C, NDC80.