In vitro translation of mRNAs that are in their native ribonucleoprotein complexes

mRNA is bound to a complex network of hundreds of RNA-binding proteins (RBPs) which constitute the mature ribonucleoprotein (mRNP). Such a complex particle is initially scaffolded in the nucleus and stays associated throughout mRNA's journey to the cytoplasm, where it participates in translation. However, due to the size, complexity and variability of the mRNP, it remains technically challenging to assess its impact on translation. By designing a novel in vitro translational assay, we have been able to compare the translational efficiency of reporter mRNAs that are, or are not, associated with their cognate RBPs. This showed the strong impact of these RBPs on translational efficiency, and revealed intrinsic variations according to the structure of both the mRNA and its nuclear history, e.g. the use of intron-containing mRNA constructs showed that splicing strongly enhanced translation. The present study shows that nuclear and cytoplasmic gene expression steps in vitro are coupled in eukaryotes and this is determined from the very birth of the mRNA in the nucleus by a network of hundreds of RBPs. * CFPS, : cell-free protein synthesis system; CMV, : cytomegalovirus; eIF, : eukaryote initiation factor; EJC, : exon junction complex; EMCV, : encephalomyocarditis virus; GAPDH, : glyceraldehyde-3-phosphate dehydrogenase; glo, : β-globin; HCV, : hepatitis C virus; IRES, : internal ribosome entry site; luc, : luciferase; mRNP, : mature RNP; qPCR, : quantitative PCR; RBP, : RNA-binding protein; RNP, : ribonucleoprotein; RRL, : rabbit reticulocyte lysate; RT, : reverse transcription; Su, : post-ribosomal supernatant; TPI, : triose phosphate isomerase; uRRL, : untreated RRL