Recruitment Sequencing (Rec-Seq) allows transcriptome-wide analysis of early steps of translation initiation in a purified system.

(A) Overview of steps in the Rec-Seq method. 48S ribosomal pre-initiation complexes (PICs) are assembled in vitro from purified S. cerevisiae components. 48S PICs are treated with RNase I to digest unprotected mRNA and then isolated using sucrose density gradient ultracentrifugation. Ribosome-protected mRNA fragments (RPFs) are used to construct a sequencing library. (B-D) Metagene plots of RPF distributions over 150 nt windows (−50 to +100 nt) on all mRNAs aligned to their main AUG start codons for no Ded1 (B); 100 nM Ded1 (C), or 500 nM Ded1 (D). RPF lengths are shown on the Y axis and the position of the 5’ends of the RPFs relative to the main start codon are shown on the X axis. A 5’end located 12 nt from the start codon is expected for 48S PICs with the AUG in the P site of the 40S subunit (Wagner et al. 2020). The color scale shows RPF density. All reads for 3 replicates for each condition were combined. (E, F) RPFs for the 0, 100 and 500 nM Ded1 experiments for previously identified Ded1 hypo-dependent (OST3, E) and hyper-dependent (HOR7, F) mRNAs. The position of the main CDS is shown in cyan and the −3 to −1 and +4 context nucleotides surrounding the main AUG are shown in brick red text. mRNA sequencing reads are also shown below each set of tracks (gray). The Integrated Genome Viewer (IGV, Broad Institute) was used to display RPF and mRNA reads, with RPF and nucleotide (nt) scales indicated on top of each panel. For these and all other gene browser views, the RPFs are plotted to their predicted P-site positions, and the mRNA reads are plotted to their first position from their transcript 5’ends.

Ded1 promotes the recruitment of a group of 1000 mRNAs.

(A-C) Scatterplots of normalized read densities on the main start codons of mRNAs (mRPF, number of RPFs mapping to the main AUG normalized by the geometric means of the spike-in internal control RPFs) for 3052 mRNAs with > 90 total reads in 9 samples (3 replicates each for 0, 100 and 500 nM Ded1) for 100 versus 0 nM Ded1 (A), 500 versus 0 nM Ded1 (B), or 500 versus 100 nM Ded1 (C). mRNAs with significantly changed recruitment were defined as those with false discovery rates (FDR) < 0.05 and RPF changes (ΔRPF) of > 2 for increased or < 0.5 for decreased are indicated in red or dark blue, respectively. The number of significantly changed mRNAs are indicated in red (increased) and blue (decreased). (D and E) Overlaps between the mRNAs displaying significantly increased (D) or significantly reduced (E) mRPFs between 100 and 500 nM Ded1 conditions. P-values for the overlap between each group were calculated using an online tool (http://nemates.org/MA/progs/overlap_stats.html). (F) Box plot analysis of the distribution and median of log2 change in mRPF between 0 and 100 nM Ded1 (ΔmRPF100/0 nM Ded1, orange) or 0 and 500 nM Ded1 (ΔmRPF500/0 nM Ded1, cyan) for all 3052 mRNAs (cols. 1-2), and for the following 6 groups of mRNAs: 1006 or 911 for which mRPFs were significantly up in 500 or 100 nM Ded1, respectively, relative to no Ded1 (cols. 3-6, for red dots in B and A); 793 mRNAs for which mRPFs were significantly up with both 100 and 500 nM Ded1 (cols. 7-8, for the overlapping mRNAs in D); 182 and 71 mRNAs for which mRPFs were significantly down in 500 or 100 nM Ded1 relative to no Ded1 (cols. 9-12, blue dots in B and A); and 48 mRNAs for which mRPFs were significantly down with both 100 and 500 nM Ded1 (cols.13-14, for the overlaps in E). The blue horizontal line shows no change in mRPFs (ΔmRPF = 1). (G) Heat-map analysis of the recruitment efficiency changes ΔRE100/0 nM Ded1, ΔRE500/0 nM Ded1 and ΔRE500/100 nM Ded1 for the 3052 mRNAs described in A-C, ordered by rank of ΔRE100/0 nM Ded1 from most increased (top) to most decreased (bottom) using the R heatmap.2 function. (H) The distributions of all observed mRNAs across 10 bins of increasing RE values at each concentration of Ded1 (0, 100, 500 nM). (I) Boxplot analysis of ΔRE500/0 nM Ded1 for all 3052 observed mRNAs binned by RE in the absence of Ded1 (RE-Ded1) from the lowest to the highest.

Ded1 promotes the recruitment of mRNAs with long or structured 5’UTRs.

(A) Line plot analysis of average recruitment efficiency (RE) for the 2804 mRNAs observed in the RecSeq experiments that have annotated 5’UTRs binned by 5’UTR length at 0, 100 and 500 nM Ded1. Average RE values were determined from the RE values of all mRNAs in each bin. (B-D) Box plot analyses of the recruitment efficiency changes (ΔRE) between each of two different conditions for the same mRNA bins as in (A) for ΔRE100/0nM_Ded1 (B), ΔRE500/0nM_Ded1 (C), or ΔRE500/100nM_Ded1 (D). (E) Line plot analysis of average RE values for the 1874 mRNAs observed in the RecSeq experiments with reported PARS scores binned according to Max30 PARS scores from the lowest to highest. PARS scores were determined by Kertesz et al. (2010) and Max30 and other PARS score intervals were calculated as described in Sen et al. (2015). (F-H) Box plot analyses for the same mRNA bins as in (E) for ΔRE100/0nM_Ded1 (F), ΔRE500/0nM_Ded1 (G), or ΔRE500/100nM_Ded1 (H). (I) Line plot analysis of average REs for all 3052 mRNAs divided into six equal-sized bins according to CDS lengths from the shortest to the longest for 0, 100 and 500 nM Ded1. (J-L) Box plot analysis for the same mRNA bins as in (I) for ΔRE100/0nM_Ded1 (J), ΔRE500/0nM_Ded1 (K), or ΔRE500/100nM_Ded1 (L). All bins contain an equal number of mRNAs.

Ded1 dependencies observed in Rec-Seq experiments correlate strongly with in vivo results from previous ribosome profiling of the ded1-cs mutant.

(A-B) Scatterplots comparing Rec-Seq input RNA reads and ribosome profiling mRNA reads of WT (A) or ded1-cs (B) strains. (C) Scatterplot comparing ribosome profiling mRNA reads between WT and ded1-cs strains. (D-F) Scatterplots comparing 48S PIC RPFs from Rec-Seq to 80S RPFs from ribosome profiling for the 3035 mRNAs that passed the significance cutoffs in both experiments for Rec-Seq at 0 nM Ded1 versus ribosome profiling of the ded1-cs mutant (D) or Rec-Seq at 100 nM (E) or 500 nM (F) Ded1 versus ribosome profiling of the WT strain. (G-H) Scatterplots comparing changes in translational efficiency (ΔTE) from ribosome profiling of the ded1-cs mutant versus WT to ΔRE0/100nM_Ded1 (G) or ΔRE0/500nM Ded1 (H) values from Rec-Seq. (I-K) Boxplot analysis of 5’UTR lengths (I), Max30 PARS (J), or Total PARS (K) for mRNAs with annotated 5’UTR lengths or PARS scores that showed significantly decreased (Down) or increased (Up) REs in 100 nM or 500 nM Ded1 versus no Ded1, or significantly increased (Up) or decreased (Down) TEs in the ded1-cs mutant versus WT. (L-M) Overlaps between mRNAs identified by Rec-Seq at 100 nM Ded1 (L) or 500 nM Ded1 (M) with significantly increased REs versus no Ded1 in Rec-Seq and mRNAs with significantly increased TEs in ribosome profiling of the ded1-cs mutant versus WT. P-values for the overlaps were calculated as described in Fig. 2D-E. Group X and Y mRNAs in (L) are those in common between the two groups being compared (X) or those found exclusively in the set of 1002 mRNAs with significantly increased RE in 500 nM Ded1 versus no Ded1 in Rec-Seq (Y). (N-O) Boxplot analysis of log2 ΔRE500/0nM_Ded1 (N) or log2 ΔTEded1-ts- (O) values for all 3035 mRNAs observed in both Rec-Seq and ribosome profiling, the 1002 mRNAs with significantly increased recruitment with 500 nM Ded1 versus no Ded1 (of L), the 319 mRNAs in group X (of L), and the 683 mRNAs in group Y (of L).

Ded1 increases leaky scanning of main start codons

(A) The ratio of the total RPFs internal to the CDS in mRNAs, excluding the main start codon, to the total RPFs for the full CDS including the main start codon (iRPF/cdsRPF ratio) for each of the three replicates at 0, 100 and 500 nM Ded1. iRPFs were counted from the +9 position of the main AUG to the stop codon, while the cdsRPFs were counted from the start codon to the stop codon of the CDS. The average of the three replicates is indicated by the colored bars (red, green and blue for 0, 100 and 500 nM Ded1, respectively). (B-D) Metagene plots showing RPF density distribution on all mRNAs aligned to their main AUGs as in Fig. 1B-D for 0 (B), 100 (C) and 500 nM Ded1 (D), with color scales adjusted to show internal RPFs (iRPFs). (E-H) 48S PIC RPFs and input mRNA reads on four selected mRNAs that showed elevated internal ribosome occupancies in 100 and 500 nM Ded1 conditions. The −3 to −1 and +4 context nucleotides surrounding the main AUG, iAUG or upstream AUG (uAUG) are indicated in brick red text. (I-J) Scatterplots comparing log2 iRPF between 100 (I) or 500 nM Ded1 (J) to 0 nM Ded1 as described in Fig. 2A-B for the same group of 3052 mRNAs. Red dots show mRNAs that meet the significance cutoff for increases in iRPF (FDR < 0.05, >2-fold increase in iRPFs) and blue dots show mRNAs that meet the significance cutoff for decreases in iRPF (FDR < 0.05, <0.5-fold decrease in iRPFs). (K) Box plot comparing mRPFs and iRPFs between 500 and 0 nM Ded1 for the 182 mRNAs that showed significantly reduced mRPFs with 500 nM Ded1 versus no Ded1. (L) Overlaps between mRNAs with significantly elevated iRPFs (orange), significantly reduced mRPFs (green) or significantly increased mRPFs (blue) with 500 nM Ded1 relative to 0 nM Ded1. The Venn diagram was generated and p-values calculated as in Fig. 2D. (M) Scatter plot comparing mRPFs with 500 nM versus 0 nM Ded1. The 125 mRNAs that showed significantly increased iRPFs and significantly decreased mRPFs in 500 nM Ded1 relative to 0 nM Ded1 are labeled by black dots. Among this set, the mRNAs for which the iRPFs increase by at least 50% of the decrease in mRPFs are labeled by yellow circles, to indicate the 84 mRNAs for which the decrease in mRPFs could be responsible for the increase in iRPFs. (N) Boxplot comparing mRPFs and iRPFs between 500 nM and 0 nM Ded1 for the 1006 mRNAs that showed significantly increased mRPFs. (O) Line plot analysis of average RRO (relative ribosome occupancy; iRPF/cdsRPF ratio; left axis) and ΔRRO500/0 nM Ded1 (right axis) for 2441 of 3052 mRNAs in J with 5’UTR length >5 nt binned by main AUG context scores from lowest to highest. (P) Boxplot analysis of Start30, Plus15, Plus30 and Plus45 PARS scores for mRNAs with RROiRPF/cdsRPF < 0.5 (orange) or ≥ 0.5 with 500 nM Ded1.

Ded1 modestly promotes readthrough of start codons in 5’ UTRs of mRNAs.

(A) The ratios of RPFs in 5’UTRs to RPFs on main start codons (uRPF/mRPF ratios) for each of the three replicates with 0 (blue), 100 (magenta) or 500 (green) nM Ded1. The 5’UTR RPFs (uRPFs) were counted from the 5’end of the mRNA to the −5 position relative to the main AUG. The mean of the three replicates is indicated by the colored bars. uRPFs on GCN4 mRNA were analyzed separately (Fig. 6-S1), because 42% and 57% of all uRPFs were mapped to the GCN4 5’UTR in assays with 100 and 500 nM Ded1, respectively. (B-C) Scatterplots comparing uRPFs in the presence of either 100 nM (B) or 500 nM (C) Ded1 versus 0 nM Ded1. mRNAs with significantly increased or decreased uRPFs in the presence of Ded1 are indicated in red or dark blue dots, respectively. The criteria used for significance were FDR < 0.05 and a greater than 2-fold increase or decrease. Yellow circles denote the very few mRNAs whose uRPF read number decreases by more than 50% of the increases in their mRPF reads, indicating a clear reciprocal relationship between the decrease in uRPFs and the increase in mRPFs. (D) Box plot analysis of mRPF and uRPF read numbers for the 257 mRNAs that had both uRPFs and mRPFs ≥ 2 reads averaged over all assays conducted at 0, 100 and 500 nM Ded1 concentrations. Unlogged median RPF numbers are labeled under each box.

eIF4A enhances the recruitment of almost all mRNAs

(A) Scatterplots of normalized read densities mapped to main AUGs (mRPFs) with 5000 nM versus 500 nM eIF4A for the 2809 mRNAs with ≥ 8 total reads in 4 samples (2 replicates each for 5000 and 500 nM eIF4A). Red and blue dots show mRNAs with mRPFs significantly increased or decreased, respectively, at 5000 versus 500 nM eIF4A. The criteria for significance were FDR < 0.05 and a > 2-fold change in RPFs. (B) Boxplot comparing ΔRE5000/500 nM eIF4A (grey) to ΔRE100/0 nM Ded1 and ΔRE500/0 nM Ded1 (cyan) for the 2698 mRNAs that passed the cutoff mentioned in (A) in both experiments. (C) Boxplot of ΔRE5000/500 nM eIF4A for the 2538 of all 2809 mRNAs in (A) that have annotated 5’UTRs, divided into six equal sized bins according to 5’UTR lengths from the shortest to the longest. (D) Similar to (C), but for the 1708 of all 2809 mRNAs in (A) with annotated PARS scores, binned by Max30 PARS from lowest to highest. (E-G) Line plots of mean log2 RE changes, ΔRE5000/500 nM eIF4A (green), ΔRE100/0 nM Ded1 (magenta), and ΔRE500/0 nM Ded1 (blue), for mRNAs divided into six equal sized bins according to the specific mRNA features of 5’UTR length (E), Max30 PARS score (F), or Total PARS score (G). (H-K) RPFs assembled with 500 or 5000 nM eIF4A and input mRNA reads for mRNAs shown previously to be hypo-dependent on Ded1 (RPC10, RPL41A, HOR7) or hyper-dependent on Ded1(PMA1) by in vivo ribosome profiling experiments in the ded1-cs mutant.

A schematic diagram of mRNA recruitment in translation initiation.

The cap-binding protein complex eIF4F, consisting of eIF4E, eIF4G and eIF4A subunits attaches to the cap at the mRNA 5’ end. The 43S PIC, which includes the 40S ribosomal subunit, eIFs 1, 1A, 3 and 5 and the eIF2•GTP•Met-tRNAi ternary complex, binds to the 5’ end of mRNA with the aid of the eIF4F complex and eIF4B and scans downstream in search of the start codon. A stable 48S-PIC is formed when a start codon is recognized.

Internal spike-in normalization controls using preformed 48S PICs on non-native mRNAs.

(A) Steps in applying 48S-PIC spike-in controls for data normalization. Firefly and Renilla luciferase mRNAs (FLUC and RLUC) were in vitro transcribed from PCR-amplified gene templates containing T7 promoters and enzymatically capped using previously described protocols (Acker et al. 2007). The 48S PICs formed on the spike-in mRNAs were prepared separately from the testing samples in a recruitment reaction followed by RNase I treatment to digest unprotected mRNA. A constant amount of spike-in 48S PICs were resolved by sedimentation through the same sucrose gradients containing the experimental samples. The RPFs from these spike-in PICs served as internal controls for losses occurring during library construction and allowed data normalization across experimental samples. For deep sequencing data processing, non-coding RNA reads were removed and the remaining RPFs were aligned to spike-in mRNA sequences for calculating sample size factors and also aligned to the yeast genome to produce RPF counts on all yeast mRNAs. The RPF counts for all mRNAs in the genome were applied to DESeq2 using sample size factors calculated from the geometric means of spike-in mRNA RPFs mapped to FLUC/RLUC main AUGs and a number of internal AUGs. Recruitment efficiency (RE) values were calculated by normalizing the size-factor corrected RPFs to input mRNA densities. (B-D) Scatterplots of FLUC and RLUC RPF reads at the main and internal AUGs, in the first two of three replicate samples for each condition. The green diagonal line is for 1:1 ratio and the Spearman correlation coefficients (r) were calculated using “cor.test” function in R using option “method = spearman.”

Transcriptome-wide reproducibility of RPFs among replicates

(A-I) Scatterplots of log2 main AUG ribosome occupancies (normalized by the geometric means of RPF reads on FLUC and RLUC spike-in mRNAs) for comparing each of two of the three replicates in different conditions, 0 nM Ded1 (A, D and G), 100 nM Ded1 (B, E and H) and 500 nM Ded1 (C, F and I) for all mRNAs with ≥ 90 total RPF reads in the 9 samples combined. Spearman correlation coefficients (r) were calculated as in Fig. 1-S2B-D.

RPFs on main AUGs are consistent among replicates.

(A) Box plot analysis of normalized RPF reads for 3052 mRNAs with > 90 total reads in 9 samples (each of 3 replicates for 0, 100 and 500 nM Ded1). (B-I) 48S PIC-protected fragments (RPFs) and input mRNA reads for 3 replicates of each of the 3 conditions (0, 100 and 500 nM Ded1) on 8 example mRNAs across 3’ portions of the 5’UTR and 5’ portions of the CDS regions flanking the main AUGs. RPF and nt scales are shown in the top right corner of each panel. The position of the main CDS and the −3 to −1 and +4 context nucleotides surrounding the main AUG are labeled as in Fig. 1E-F. mRNAs shown are (B) NPT1; (C) PMA1; (D) SFT2; (E) ICE2; (F) AIM41; (G) RCP10; (H) RPL41A; and (I) RPL16A.

Ded1 preferentially stimulates 48S PIC formation on structured mRNAs.

(A, B) Line plot analysis of log2 average REs at 0, 100 and 500 nM Ded1for mRNAs binned according to total PARS scores (A) or the sum of PARS scores of the 30 nucleotides surrounding the start codon (for mRNAs with a 5’UTR of ≥15 nt) (Start30 PARS, B). (C, D) Line plot analysis of log2 average ΔREs for 100/0 nM, 500/0 nM and 500/100 nM Ded1 for the 2035 mRNAs observed in RecSeq that have annotated PARS scores binned according to Total PARS (C) or Start30 PARS (D) scores from the lowest to highest. (E-F) Box plots comparing 5’UTR or CDS lengths for the 4694 yeast mRNAs that have annotated 5’UTRs (Pelechano et al. 2013) for CDS lengths plotted for mRNAs binned by 5’UTR length from the shortest to the longest (E), or 5’UTR lengths plotted for mRNAs binned by CDS lengths from shortest to longest (F). All bins contain equal numbers of mRNAs.

Ded1 promotes the recruitment of mRNAs with long 5’UTRs independent of CDS lengths.

(A-C) Similar to Fig. 3C, but for mRNAs with different CDS length ranges, short (A, 78 – 804 nt, n=936), medium (B, 807-1389 nt, n=934) and long (C, 1392-7242 nt, n=934). (D-F) Similar to Fig. 3K, but for mRNAs with different 5’UTR length ranges, short (D, 1-31 nt, n=958), medium (E, 32-60 nt, n=925) and long (C, 1392-7242nt, n=921). (G-I) Box plot analysis of mRNA 5’UTR lengths for the same groups of mRNAs as in panels D-F, respectively.

Ded1 has little effect on discriminating main AUG context scores.

(A) Line plot analysis of average REs for the 2768 observed in RecSeq that have annotated 5’UTR lengths >5 nt, divided into six equal-sized bins according to context scores from lowest to the highest at 0, 100 and 500 nM Ded1. (B-C) Box-plot analyses of ΔRE100/0nM Ded1 (B) and ΔRE500/0nM Ded1 (C) values for the same mRNA bins as in (A).

Supporting evidence that Ded1 promotes leaky scanning of the main start codon.

(A) Schematic of a 43S PIC leaky scanning through the main AUG and assembling at an internal AUG in the presence of Ded1. (B) Evidence that mRNAs whose recruitment is inhibited by 500 nM Ded1 in Rec-Seq assays generally assemble 48S PICs very efficiently without Ded1. Boxplot analysis of REs of groups of mRNAs at 0 nM (cyan) or 500 nM (yellow) Ded1 for all 3052 mRNAs observed in Rec-Seq, the 1006 mRNAs with significantly increased mRPFs at 500 nM Ded1 versus no Ded1 (Fig. 2B, red dots); and the 182 mRNAs with significantly decreased mRPFs with 500 nM Ded1 versus no Ded1 (Fig. 2B, blue dots).

mRNAs with reduced RE at 500 nM Ded1 versus no Ded1 in Rec-Seq assays tend to show increased relative TE in the ded1-cs mutant versus WT in ribosome profiling experiments.

Boxplot analysis of ΔTEded1-cs for the groups of mRNAs described in Figure 5L-M: The 182 mRNAs with mRPFs significantly reduced at 500 nM Ded1 versus no Ded1 (cyan); the 125 mRNAs with significantly decreased mRPFs and increased iRPFs with 500 nM Ded1 versus no Ded1 (magenta); the 84 mRNAs from the previous set that had an increase in iRPFs that was at least 50% the of the decrease in mRPFs (yellow circles in Fig. 5M; orange).

Approximately half of all uRPFs map to the GCN4 5’UTR.

Percentages of all uRPFs mapped to the GCN4 5’UTR for each of the three replicates with 0 (blue), 100 (magenta), or 500 (green) nM Ded1 concentrations. Colored bars indicate the means of the replicates.

Oligonucleotides used for Spike-in mRNA template amplification