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. 2020 Aug 20;79(4):546–560.e7. doi: 10.1016/j.molcel.2020.06.004

Figure 3.

Figure 3

Sel-TCP-Seq Detects Staged Dissociation of eIFs from Translation Initiation Complexes

(A) Human unselected 40S FP versus eIF3b::40S FP counts per mRNA. r, Pearson correlation coefficient.

(B) As (A) but for yeast unselected 40S versus eIF3a::40S.

(C) As (A) but for yeast unselected 40S versus eIF2β::40S.

(D) Human eIF3b::40S FP and unselected 40S FP coverage aligned to annotated transcription start sites (cap; 1,046 mRNAs, excluding start codon-associated FPs). Averages of replicates 1 and 3 are shown.

(E) As (D) but for yeast eIF3a::40S and unselected 40S (198 mRNAs).

(F) As (E) but for yeast eIF2β::40S and unselected 40S (198 mRNAs).

(E and F) Averages of replicates 1 and 2 are shown.

(G) Human eIF3b::40S versus unselected 40S FP count ratios within indicated mRNA regions (n = 1,389; dashed line, median; dotted lines, first and third quartiles; ∗∗∗p < 0.001, two-sample t test). Data from human replicate 3 are shown.

(H) As (G) but for yeast eIF3a::40S versus unselected 40S FP count ratios (n = 2,096, ∗∗∗∗p < < 0.0001). Data from yeast replicate 1-1 are shown.

(I) As (H) but for yeast eIF2β::40S versus unselected 40S FP count ratios (n = 1,734). Data from yeast replicate 1-3 are shown.

(J) Enrichment of tRNA read counts in human eIF3b::40S versus an unselected 40S library. Averages of human replicates 1 and 3 are shown.

(K) As (J) but for yeast eIF3a::40S and eIF2β::40S versus the corresponding unselected 40S libraries. Averages of replicates 1 and 2 are shown.

(L) Differences in FOI (indicated below the x axis) persistence upon initiation complex arrival at start codons. The ratio of FOI::40S FP counts in the 5′ UTR versus start codon regions of 1,218 mRNAs was divided by the equivalent ratio of unselected 40S FP counts. Both FOI::40S complexes redistribute to 5′ UTRs relative to the unselected 40S (∗∗∗∗p < < 0.0001, one-sample t test), but this is more pronounced for eIF2β than eIF3a (∗∗p < 0.005, two-sample t test). Data from yeast replicate 1 are shown.

(M) Human unselected 80S FP and eIF3b::80S FP coverage frequency at the beginning of the CDS of 10,778 mRNAs. Averages of replicates 1 and 3 are plotted.

Error bars in (D)–(F), (J), (K), and (M) represent ± standard deviation.