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I. Boussaid et al.
Discussion
RP depletion has been recognized as a principal cause of erythroid hypoplasia either in acquired del(5q) MDS or DBA.2,36 The specific impairment of the erythroid lineage has been linked to a decreased representation of GATA1 transcript on polysomes under conditions of an RPS19, RPL5, RPL11 or RPS14 haploinsufficiency.6,11 Our current results indicate that in addition to a reduction in GATA1 mRNA on polysomes, translation as a whole is selective at the expense of erythroid transcripts including globin genes under conditions of low ribosome availability. Consistent with the previous findings of Yang et al.,8 we have observed in our present study that low globin gene trans- lation may account for a disequilibrium in the heme-glo- bin balance, leading to reactive oxygen species (ROS) pro- duction and cell death.
Translation efficiency is thought to depend on the ther- modynamic properties of a given transcript.23,37 For exam- ple, the presence of an IRES in the BAG1 or CSDE1 mRNAs or the length of 5’UTR of the BCAT1 transcript have been implicated in disrupted translation when RPL11 or RPS19 is haploinsufficient.38,39 The structure of the 5’UTR in GATA1 mRNA has been associated with its translation downregulation in the context of RPS19 hap- loinsufficiency. However, depending on which tran- scripts were compared with GATA1 mRNA, its 5’UTR may be considered to be either highly structured or unstructured.6,12 Our visualization of the thermodynamic landscape allowed us to establish that, in comparison to all other human 5’UTR sequences, the GATA1 5’UTR is short and unstructured. Our global investigation of all the determinants of translation selectivity in UT-7/EPO and K562 shRPS14 cell line models identified that short mRNAs with a high CAI, a highly structured and short 3’UTR, and to a far lesser extent transcripts with a struc- tured 5’UTR, were specifically less translated. GATA1 mRNA is indeed a short transcript with high CAI, highly structured 3’UTR, but a less structured 5’UTR. We con- firmed our present findings using published ribosome profiling data of shRPS19/shRPL5 human primary ery- throblasts and proteome data for shRPS15 lympho- cytes.12,34 RPS rather than RPL targeting has been shown to impair the translation of mRNAs with a structured 5’UTR, highlighting the crucial role of the 40S subunit in the initiation and scanning of the 5’UTR.40 Hence, the rules of translation selectivity have been shown to be conserved across the different models of an RP deficien- cy, demonstrating that translation selectivity has a stronger association with a decrease in the cellular ribo- some content than a defect in one particular RP. Interestingly, proteins whose transcripts display most of these characteristics are accumulated during normal ery- thropoiesis suggesting that such a combination of param- eters may allow the expression of a selected proteome in a short time. Consistent with our current results, a high CAI has previously been reported to confer a high elon- gation speed and a long mRNA half-life, whereas a low CAI has been associated with mRNA decay through the slowing of translation elongation.28-30 The 3’UTR struc- ture and length are also associated with translation repression and decay.32,33 In our current experiments, under limited ribosome availability conditions, we did not observe any decay modifications linked to these two features. Further experiments are thus required to inves-
tigate the interplay between CDS and UTR and its role in the control of mRNA stability and translation.
Our current gene expression analysis highlighted path- ways involved in cell cycle, proliferation, DNA repair and RNA processing among the transcripts with the highest TE under conditions of reduced translation (Figure 2D). Notably, however, the preferential translation of these transcripts in the context of the global diminution of trans- lation rate (Figure 1J-I) remains an inefficient mechanism of rescuing the cells from death. Understanding these processes may optimize gene expression in some diseases.
Khajuria et al.12 previously reported that transcripts with unstructured 5’UTR were translated more under normal conditions and more impacted by low ribosome availabil- ity than mRNAs with a structured 5’UTR. Consistently, we found in our current analyses that the most expressed proteins during normal erythropoiesis are encoded by transcripts with an unstructured 5’UTR, and often with an unstructured 3’UTR (Figure 7D). However, many tran- scripts with a structured 3’UTR that are impacted under conditions of low ribosome availability also encode ery- throid proteins under normal conditions. Hence, RNA binding proteins and miRNA that target the 3’ end of tran- scripts may play a role in translation selectivity. Other studies have demonstrated the contribution of the 3’UTR length to the regulation of translation.21,41 Highly translated mRNAs have the ability to form a loop through interac- tions between polyA binding proteins and initiation fac- tors that brings the 5’ and 3’ ends into communication.42 It has also been suggested that ribosomes may move through the 3’UTR to support the recycling and re-initia- tion of another loop of translation at the 5’UTR.21,43 It would be advantageous for the recycling re-initiation process to have a small distance between the 3’ and 5’ ends.44 In normal conditions, a short and structured 3’UTR may contribute to an increased recycling and a higher translation efficiency by reducing the distance between 3’ and 5’ ends. In agreement with this hypothesis, recent evi- dence has demonstrated that the depletion of the ribo- some recycling/re-initiation protein ABCE1 tended to arrest ribosomes on transcripts with a short and highly structured 3’UTR.45,46 These transcripts, which have simi- lar characteristics to those we found to be preferentially translated during normal erythropoiesis, could be favored by the recycling/re-initiation process. Under conditions of RPS14, RPL5 and RPS19 downregulation, the translation of mRNAs with a short and structured 3’UTR was found to be decreased, suggesting that re-initiation could no longer occur normally and that terminating ribosomes should be released to feed the cellular pool. The overex- pression of ribosome rescue factors PELO/HBS1L in RPS19 haplo-insufficient K562 cell line, was reported to restore the hemoglobin levels.35 Whether such a mecha- nism may compensate for the loss of ribosomes in the dis- order caused by an RPS14 haplo-insufficiency will require further investigation.
Several prior yeast and human studies in which the RP genes RPS19, RPL5, RACK1 or RPS26 were mutated or deleted, have also reported that the shortest transcripts were less present on polysomes under conditions of low ribosome availability.47 This is in sharp contrast to obser- vations under normal conditions in a wide range of eukaryotic organisms, in which the shortest transcripts are more efficiently translated than the longest mRNAs.48,49 The translation initiation rate, density of
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haematologica | 2021; 106(3)