Figure 5. Pro-rich genes are preferential translational targets of EPRS.

(A) Schematic for RNA-Seq and polysome profiling-Seq (polysome-Seq) procedure in Halo treated NIH/3T3 mouse fibroblasts. Polysome-free fractions include free mRNP, 40S ribosome subunit, 60S ribosome subunit. Light polysome fractions include 80S monosome, disome, trisome, and tetrasome. Heavy polysome fractions include polysome fractions with ≥5 ribosomes.

(B) Differentially expressed genes identified by RNA-Seq and polysome-Seq are indicated by dot plot. Translation efficiency (TE) is indicated by the ratio of heavy polysome and polysome-free fraction. The colored dots indicate statistically significantly changed genes identified by either RNA-Seq or polysome-Seq. The genes with P_adj<0.05 in either of three different groups (ratio of Halo vs. Veh. treated samples for total RNA, non-polysome, and heavy polysome) were considered as significantly changed genes. All significantly changed genes were divided into four areas based on log₂FC of total mRNAs and heavy polysome mRNAs. Data were submitted to GEO database (GSE136838).

(C) Translationally dysregulated genes are indicated by overlapping changed genes in the same area of heavy polysome (B) and light polysome (Online Figure VIA). Genes decreased (Area 1) or increased (Area 3) at translation and steady-state mRNA levels are shown.

(D) A majority of genes show a synergistic change at the mRNA and translational levels after EPRS inhibition by Halo. The number of genes is shown with changes at both translation efficiency (the ratio of heavy/light polysome to polysome-free fraction) and steady-state mRNA levels in all four areas.

(E) KEGG signaling pathway analyses indicate that ECM-receptor interaction and ribosome biogenesis are the top enriched pathways in Area 1 and Area 3, respectively. DAVID Bioinformatics Resources 6.8 was used.

(F) PP motif analyses suggest that there are more PP motif-bearing genes in the translationally decreased gene cluster (Area 1) compared to the translationally increased gene cluster (Area 3).

(G) Distribution of genes according to the number of PP motifs (0, 1, 2, 3, ≥4) indicate that the genes in Area 1 contain more PP motifs compared to genes in Area 3.

(H) Frequency of PP motif normalized by protein length or gene number is significantly higher in Area 1 compared to Area 3.

Comparisons were performed by Chi-Square test for gene number counts for F, G and non-parametric unpaired Mann-Whitney test for quantification of PP motif frequency for H.