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. 2020 Jul 20;9:e56523. doi: 10.7554/eLife.56523

Figure 5. DAZL enhances translation of its targets in undifferentiated spermatogonia.

(A) Schematic of synchronization of spermatogenesis by WIN 18,446 to enrich for undifferentiated spermatogonia for RiboTag IP and sequencing experiments. (B) Schematic of the RiboTag allele. The Rpl22 locus carries a floxed exon 4, which is expressed in the absence of recombination, followed by an engineered exon four that encodes an HA tag before the stop codon. Recombination via the Ddx4-Cre allele removes the floxed exon four and allows for expression of the exon four that encodes the HA tag specifically in germ cells. The germ cells’ ribosomes can then be immunoprecipitated via the HA tag. (C) Translational efficiency of DAZL targets compared with all nontargets from undifferentiated spermatogonia (two-sided Mann-Whitney U test). (D) Translational efficiency of DAZL targets compared with nontarget subset datasets that were sampled to match transcript abundance (TPM) and CDS length in undifferentiated spermatogonia (two-sided Mann-Whitney U test). (E) Adjusted translational efficiency in undifferentiated spermatogonia (two-sided Mann-Whitney U test), calculated from a multiple log-linear regression model that included transcript abundance (TPM), CDS length, 3' UTR length, codon usage (Codon Adaptiveness Index, CAI), and DAZL binding as variables. Adjusted translational efficiency was calculated by subtracting the contributions (as calculated by the model) of transcript abundance, CDS length, 3' UTR length, and codon usage from biochemically measured translational efficiency values. ****, p<0.0001.

Figure 5—source data 1. Translational efficiencies for genes expressed in undifferentiated spermatogonia.

Figure 5.

Figure 5—figure supplement 1. Translational efficiency in undifferentiated spermatogonia.

Figure 5—figure supplement 1.

(A) Replication-dependent histones show reduced translational efficiency compared with the average transcript in undifferentiated spermatogonia. This is expected, as the undifferentiated spermatogonia here were not synchronized for S phase, when these histones are robustly translated (two-sided Mann-Whitney U test). (B) DAZL targets are distinct from nontargets in variables known to correlate with translational efficiency: transcript abundance, CDS length, 3' UTR length, and codon usage (CAI) (two-sided Kolmogorov-Smirnov tests). (C) Relationships between translational efficiency and variables known to correlate with translation efficiency: transcript abundance, CDS length, 3' UTR length, and codon usage. ****, p<0.0001.