RESUMEN
We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S pre-initiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5'-untranslated regions (5'UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5'UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5'UTRs.
RESUMEN
We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5'-untranslated regions (5'UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5'UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5'UTRs.