eIF4E-independent translation is largely eIF3d-dependent
We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3D via its cap-binding pocket. eIF3D then enables these mRNAs to be efficiently translated due to its cap-binding activity. Overall design: To see which transcripts are translated in an eIF4E-independent manner, we performed ribosome profiling in HeLa wt cells overexpressing either the empty vector or constitutively-active form of 4E-BP1. The experiment was performed in duplicates. Ribosome-protected footprints as well as total RNA were sequenced. To see if resistant transcripts are released from eIF4E upon 4E-BP1 overexpression, we performed RIP experiment with antibodies specific to eIF4E1. The IP-ed and total input fractions were sequenced. The experiment was performed in duplicates.