Ribosome profiling analysis of Denr and Eif2d depleted cells reveals the absence of re-initiation function of EIF2D

Among the transcript features that regulate translation rate, upstream open reading frames (uORFs) have emerged as a major class of inhibitory elements which limit how many scanning ribosomes reach the start codon of the main protein coding sequence (CDS). Two processes are implicated in ensuring that CDS translation of uORF-containing transcripts can take place nevertheless: first, leaky-scanning, which involves the bypassing of uORF start codons by the scanning 40S ribosome, and second, re-initiation (REI), which allows the 40S subunit of the ribosome that terminated on the uORF stop codon to resume scanning to reach a downstream start codon. REI has remained particularly enigmatic, as it is at odds with generally accepted principles of conventional translation initiation, termination and post-termination subunit recycling. MCTS1-DENR is the first re-initiation-specific factor that has been identified. Its homolog, EIF2D, is assumed to act in uORF REI as well, yet formal proof of EIF2D-mediated REI has remained scarce. In our study, we present our in vivo analyses of DENR and EIF2D in translation re-initiation in mammalian cells. At the transcriptome-wide scale, ribosome profiling clearly identifies DENR-dependent REI, yet disproves the assumption that EIF2D also functions as a REI-factor. Overall design: NIH3t3 cells were knocked-down by transduction with control shRNAs (scramble and a non-functional Eif2d shRNA) or shRNA targeting Denr or Eif2d. Biological replicates of each condition were harvested and flash frozen. Ribosome profiling and parallel RNA-seq were prepared and sequenced to compare the effect of Eif2d and Denr knock-down on translation efficiency.