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. 2018 Apr 10;13(4):e0193790.
doi: 10.1371/journal.pone.0193790. eCollection 2018.

Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors

Nahuel A Paolini  1 , Kat S Moore  1 , Franca M di Summa  1 , Ivo F A C Fokkema  2 , Peter A C 't Hoen  2   3 , Marieke von Lindern  1
Affiliations

Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors

Nahuel A Paolini et al. PLoS One. .

Abstract

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how tunicamycin treatment, that induces eIF2 phosphorylation, affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict the sensitivity of transcripts to altered ribosome recruitment in presence or absence of Tm. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Tm treatment induces phosphorylation of eIF2, reduces protein synthesis and selectively alters ribosome density of some transcripts.
(A) Murine erythroblasts (line 15.4) were left untreated (-) or were treated for 90 min with Tm (2.5 μg/ml). Western blots with total cell lysates were probed for phosphorylated (anti P-S51 eIF2) and total eIF2. (B/C) Erythroblast samples as in (A) were processed for polyA+ RNA sequencing (RNAseq; B) or ribosome footprinting (RPF; C). We normalized and averaged sequence reads from 3 biologically independent experiments. For RNAseq We applied a threshold of, on average, 10 reads per condition (B). For RFP reads we applied a threshold of, on average, 1 read per condition (C). A statistical interaction model indicated differential ribosome density on 484 transcripts at a FDR <01%, indicated as red dots. (D) Depiction of 2Log FC ribo reads and RNA reads. Dashed gray line indicates the area where translation follows transcription. The fold-change (FC) in ribo reads (Tm-treated average reads/untreated average reads; Tm/Untr) was plotted against the FC Tm/Untr in RNA expression. Figures are based on data presented in S2 Table.
Fig 2
Fig 2. Position and length of uORFs in the 5´UTR of transcripts subject to Tm-controlled translation.
(A, B) Top line indicates the distance in nt upstream of the main annotated start codon. The same relative size is used for transcripts with Tm-enhanced translation (A), or Tm-decreased translation (B), except for the last two transcripts for which size was condensed 2-fold as indicated by a separate size marker. The collapsed annotated protein ORF is shown by a grey interrupted box, with the protein name directly at the rightside. Numbers between brackets indicate the size of the main annotated protein in amino acids. Adjacent boxes with a fence pattern on the left of the “protein box” indicate a N-terminal extension of the protein. A fenced box at the back ground as shown for Hax1 indicates that this part of the annotated protein seems not translated. All uORF are indicated by open boxes, and the start codon is written at the start of the box including its Kozak context. The dashed line below indicates areas that are >90% conserved between mouse and man. A small cross below the start codon in a conserved area indicates that the start codon is not conserved. Conserved areas were identified by Blastn with the mouse sequence on the human transcriptome.
Fig 3
Fig 3. Web browser snapshot of the 5´UTR of Lysine methyltransferase 2E (Kmt2e) and Proline Rich Receptor Coactivator 2 (Pnrc2).
(A/B) Aggregate Ht- and CHX-stabilized RFP counts from three replicates of Tm-treated and untreated (Unt) cells are mapped to the genome and visualized in the UCSC web browser. Numbers on the right-hand side indicate maximum read counts in the respective lane. Only the start of the protein coding ORF is shown. (A) The data indicate two uORFs on the 5’UTR of Kmt2e. Black and gray arrow indicates Ht and CHX peak that maps to the start codon uORFs 1 and 2, respectively. uORF2 overlaps the start codon of Kmt2e. (B) The data indicate two uORFs. Black arrow indicates a TIS at start codon for uORF1, gray arrow indicates a TIS for uORF2.
Fig 4
Fig 4. Web browser snapshot of the 5´UTR of protein phosphatase 4 regulatory subunit 3B (Smek2) and Cold shock domain containing E1 (Csde1).
(A/B) Aggregate Ht- and CHX-stabilized RFP counts from three replicates of untreated (Untr) and Tm-treated cells were mapped to the genome and visualized in the UCSC web browser. Numbers on the right-hand side indicate maximum read counts in the respective lane. Only the start of the protein coding ORF is shown. (A) The data indicate a small uORF on the 5’UTR of Smek2 (gray box). Horizontal arrow indicates Ht and CHX peak that maps to the start codon of this uORF (B) The data indicate two uORFs that are depicted by grey boxes. Gray arrows indicate TISs at uORF start codons, fenced box indicates N-terminal extension and black arrow indicates the TIS of this extended protein.
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Grants and funding

This work was funded by the Landsteiner Foundation for Bloodtransfusion Research (LSBR) www.lsbr.nl [projects 1140 and 1239 to MvL]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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