Ribosome profiling through meiosis. (A) Time points (white lines) were taken through two overlapping time courses. Cartoon representations of meiotic stages are below. (B) A subset of staging controls. Positions of staging plots correspond to time points in (A). (C) Ribosome footprints across specific genes are shown for categories in fig. S4. Scales on the y axis are independent by gene.

A global view of protein synthesis through sporulation. (A) Ribosome footprints (RPKMs) were summed over each yeast gene (columns) for all samples except steady-state spores (rows). The summed expression of each gene over time points was normalized for the time course, and genes were subjected to clustering. Several clusters are noted by lines below the chart: 1, Mitochondrial ribosome; 2, nutrient uptake and/or amino acid biosynthesis; 3, mitochondrial function; 4, proteasome; 5, redox and energy generation; and 6, ribosome and translation machinery. Meiotic progression is indicated pictorially to the right. (Top) A cluster containing genes responsible for DNA replication, with the gene identities to the right. To the left is the average footprint density across the cluster, with time points corresponding to bulk DNA replication represented by arrows. (Bottom) A cluster of genes associated with recombination and SC formation. The bar to the left shows the timing of these events as determined by staging controls. Single asterisks identify genes analyzed in (B) and (C) and fig. S7. Double asterisks indicate that both MND1 and the overlapping ORF, YGL182C, were identified in this cluster. (B) Wild-type, ydr506cΔ, and ydr506c spo11Δ cells were induced to sporulate. At indicated times, samples were scored for nuclear division. (C) Wild-type, ylr445wΔ, and ylr445wΔ spo11Δ cells were induced to sporulate and were treated as in (B).

Widespread dynamic translational control in meiosis. (A) Log₂ mRNA and footprints (RPKM) for a region containing SPS1 and SPS2 over pooled time points (fig. S4). (B) SPS1-3HA and SPS2-FLAG cells carrying an estrogen-inducible NDT80 allele were induced to sporulate. At 6 hours, β-estradiol was added. Samples from indicated times were subjected to Western blotting (WB) and Northern blotting (NB). (C) Log₂ TE values for CLB3 and YPT1 for pooled time points (fig. S4). MI and MII are indicated by colored shading as boxes. (D) Cluster analysis of log₂ TE through meiosis for pooled categories (fig. S4) for all genes. (E) Log₂ TEs are plotted as in (C) for AMA1, RCR1, ORC1, and ZIP1. (F) Log₂ TEs are plotted as in (C) for HAC1. Below, total RNA from the original time course (see fig. S1) was subjected to Northern blotting for HAC1.

Noncanonical translation is pervasive in meiotic cells. (A) Footprints from pooled time points (see fig. S4) were mapped. The percentage of these footprints outside of known ORF annotations is plotted. (B) mRNA and ribosome occupancy profiles around YOL092W, with sense above the line for each time point and antisense below. Single asterisk denotes the sORF start site. The “AUG unit” (sORF) was annotated by the strategy shown in fig. S11. (C) The region around SAS4 is displayed as in (B), with truncated ORF start denoted by a single asterisk. (D) Ribosome occupancy profile for vegetative cells in exponential growth phase and meiotic cells over the leader of ACB1. (E) For pooled time points (see fig. S4), TEs are plotted for ORFs and for leaders (see SOM for a discussion of leader TE determination) for IME1, CDC28, and PDS1. Values are normalized to the same range for both plots. (F) Correlation coefficients [determined from plots as in (E)] were determined for each gene with uORFs for leaders with only near-cognate uORFs and at least one AUG uORF. The positions of six genes that support cap-independent translation (39) are noted.

Regulated transcript extensions expose novel regulatory uORFs. (A) mRNA and ribosome occupancy profiles around SUP35. (B) ORC1 region displayed as in (A). (C) Total RNA from the original time course (see fig. S1) was subjected to Northern blotting for ORC1. (D) Analysis as in Fig. 4E for ORC1, SUP35, NDJ1, RED1, NDC80, and POP4. (E) Analysis as in Fig. 4F for genes with regulated leader extension (table S5).