Editors' ChoiceStem Cells

Translational complex for differentiation

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Science Signaling  17 Jan 2017:
Vol. 10, Issue 462, eaam7563
DOI: 10.1126/scisignal.aam7563

NAT1 interacts with a subset of eukaryotic translation initiation factors to enable expression of transcripts required for stem cell differentiation signals.

Stem cells have two critical characteristics: the ability to self-renew and the ability to differentiate. Mouse embryonic stem (mES) cells genetically deficient in Nat1, which encodes a protein with homology to eukaryotic translation initiation factor 4G (eIF4G), cannot differentiate and exhibit a phenotype similar to the “ground” state, which is characterized by cells with a uniform morphology and high abundance of proteins associated with self-renewal. Combined pharmacological inhibition of the kinases of the ERK family and the GSK3 family put wild-type mES cells into the ground state. Transcriptomic analysis showed that Nat1–/– mES cells had a profile more similar to that of wild-type mES cells that had been induced into the ground state by kinase inhibition than to wild-type mES cells that had not been exposed to the kinase inhibitors. The abundance of transcription factors associated with self renewal was similar in the Nat1–/– mES cells and in the ground state wild-type mES cells. Also similar to the ground state wild-type mES cells, phosphorylation of ERK was decreased and phosphorylation of STAT3 was increased. However, the ground state cells and the Nat1–/– mES cells differed with regard to AKT phosphorylation (lower in Nat1–/– mES cells) and GSK3 phosphorylation (lower in ground state wild-type cells). Ribosome profiling revealed 14 genes with decreased translation and 4 with increased translation in the Nat1–/– mES cells. Genes encoding MAP3K3 and SOS1, which act upstream of ERK, were among those with suppressed translation in the Nat1–/– mES. Expression of MAP3K3, but not SOS1, restored differentiation capacity to the cells, on the basis of morphological analysis criteria, decreased abundance of transcripts that encode pluripotency markers, and increased abundance of transcripts encoding proteins indicative of differentiation. Mass spectrometry analysis of proteins that coimmunoprecipitated with FLAG-tagged NAT1 or eIF4G (introduced by CRISPR/Cas9 recombination) revealed that these two proteins formed distinct translational complexes. Whereas NAT1 preferentially interacted with eIF2, FMR1, FXR1, PRRC2A, PRRC2B, and PRRC2C and not the cap-binding protein eIF4E; eIF4G bound to proteins associated with recognition or processing of poly-A tails and cap-dependent translation. This study suggests that, even under nonstressed conditions, translation is controlled by distinct complexes with distinct mRNA targets and that NAT1 mediates a form of cap-independent translation of genes necessary for stem cell differentiation.

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