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.
CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Lyon, France Inserm, Institut National de la Santé et de la Recherche Médicale, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, CIRI, Centre International de Recherche en Infectiologie, Lyon, France CNRS, Centre National de la Recherche Scientifique, UMR5308, Lyon, France.
Baptiste
B
Panthu
CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Lyon, France Inserm, Institut National de la Santé et de la Recherche Médicale, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, CIRI, Centre International de Recherche en Infectiologie, Lyon, France CNRS, Centre National de la Recherche Scientifique, UMR5308, Lyon, France.
Fabrice
F
Mure
CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Lyon, France Inserm, Institut National de la Santé et de la Recherche Médicale, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, CIRI, Centre International de Recherche en Infectiologie, Lyon, France CNRS, Centre National de la Recherche Scientifique, UMR5308, Lyon, France.
Henri
H
Gruffat
CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Lyon, France Inserm, Institut National de la Santé et de la Recherche Médicale, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, CIRI, Centre International de Recherche en Infectiologie, Lyon, France CNRS, Centre National de la Recherche Scientifique, UMR5308, Lyon, France theophile.ohlmann@ens-lyon.fr.
Theophile
T
Ohlmann
D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D000067556:Internal Ribosome Entry Sites / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
D000067556:Internal Ribosome Entry Sites; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D008156:Luciferases
RBPs; hybrid system; in vitro translation; mRNP; translation