Page 106 - 2021_03-Haematologica-web

I. Boussaid et al.
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Figure 5. Impact of each mRNA feature on the translation efficiency and transcript stability. (A) Cumulative frequency representation of translation efficiency changes for mRNAs with the highest and lowest codon adaptation index (CAI), the largest and lowest 3’UTR ∆G.base-1 or 5’UTR ∆G.base-1 and the largest and short- est lengths. (B) Cumulative frequency representation of stability changes (Log2 [FC] transcriptome) of transcripts with the highest and lowest CAI, the largest and low- est 3’UTR ∆G.base-1 or 5’UTR ∆G.base-1 and the largest and shortest lengths. Distances were calculated using the Kolmogorov-Smirnov test. Exact P-values were cal- culated.
RNA polymerase I regulation while the few downregulat- ed gene sets showed an involvement in cell proliferation and steroid biogenesis. GO term enrichment analysis of significantly impacted genes in the translatome and tran- scriptome confirmed these findings (Online Supplementary Table S3 and Online Supplementary Figure S2).
The highest and lowest expressed transcripts with an FC >1.5 or <1/1.5 in the translatome and transcriptome were subsequently selected (Figure 2E). A higher number of transcripts of the translatome varied in comparison to transcripts of the whole transcriptome (Figure 2F), with lit- tle overlap between the transcripts found to be differen- tially expressed in the translatome and in the transcrip- tome (Figure 2G). We then extended our analysis to a pub- lished dataset of DBA models targeting RPL5 or RPS19.12 Remarkably, despite differences in cell types, targeted RPs and methods, the ΔTE across the models was highly cor- related, particularly when RPS14- or RPS19-targeted cells were compared (Online Supplementary Figure S2B). Since these global expression analyses indicated uncoupling of the translatome and transcriptome, we concluded that a mechanism of selective gene expression regulation was operating at a translational level.
The codon adaptation index, coding sequence length and thermodynamic characteristics of the 3’UTR govern the translation selectivity under conditions of limited ribosome availability
Multiple mechanisms of translation regulation are dependent on the mRNA sequence and structure.19-22 We
first focused our analysis on the contribution of UTR to translation selectivity. We took advantage of the availabil- ity of thermodynamic parameters of UTR in the University of California Santa Cruz (UCSC) databases to establish the thermodynamic landscape of all the refer- enced 5’ and 3’UTR in humans.23 Secondary structures in mRNA UTR are characterized by the enthalpy ΔG (fold energy) and the length which are highly correlated (Online Supplementary Figure S3A) and the fold energy per base which relationship to the length is not linear, each of them influencing translation efficiency (Online Supplementary Figure S3B). We compared the 5’UTR and 3’UTR parame- ters of the 100 transcripts that were the less impacted or the more downregulated in the translatome and transcrip- tome (Figure 3A). The 5’UTR energy per base was similar between the up- and downregulated mRNAs in the tran- scriptome and was generally slightly stronger in the mRNAs downregulated on the polysomes (Online Supplementary Figure S3C). The 5’UTR energy per base measure provided a poor separation of the less impacted and the more downregulated transcripts of the trans- latome while the 3’UTR thermodynamic characteristics were fully distinctive (Figure 3B and C). A strong negative energy per base of the 3’UTR defining a highly structured region and its shortness characterized the transcripts with a lower presence on heavy polysomes, indicating that they were less translated under conditions of RPS14 downregulation (Figure 3C and Online Supplementary Figure S3D). By contrast, the 3’UTR thermodynamic char- acteristics of the most and the less expressed transcripts in
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haematologica | 2021; 106(3)