The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes

Abstract

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.

The RNA Helicase Ded1 from Yeast Is Associated with the Signal Recognition Particle and Is Regulated by SRP21

“…Ded1 remains attached to the mRNA during scanning by the 48S PIC, the formation of the 48S IC at the AUG start codon, and the eventual assembly of the 80S ribosomes ( Figure 10 A). This is consistent with crosslinking experiments of ribosomal RNA that show Ded1 near the mRNA entry channel [ 24 , 46 ]. The RNA-dependent ATPase activity of Ded1 is uninhibited and it is able to translocate on the mRNA with the ribosomes through rapid cycling between the “open” and “closed” conformations, and it may further stabilize the ribosome-mRNA complex during scanning, assembly, and translation [ 14 ].…”

“…Ded1 is considered a translation-initiation factor ([ 14 , 15 ] and references therein) but crosslinking studies on DDX3 show most of the interactions on the open reading frames of a subset of the mRNAs [ 76 ]. We obtained similar results with Ded1 [ 24 ]. Thus, Ded1 remains associated with the ribosomes during translation elongation, which can be seen in polysome profiles as well [ 13 ].…”

“…These glucose-depletion results are consistent with Ded1 interacting with actively translated mRNAs because there is a 50% reduction in the percentage of mRNAs recovered, and there is a significant shift to crosslinked mRNAs that encode proteins involved in metabolic shifts. In both growth conditions, the vast majority of the recovered mRNA fragments are on the coding regions, which provided further evidence that the interactions are on actively translating mRNAs [ 24 ].…”

“…SRG1 negatively regulates the transcription of the SER3 gene as capped and polyadenylated RNAs [ 44 ]. We recover fewer SRG1 RNA fragments under glucose-depletion conditions [ 24 ]. In contrast, we recover more crosslink fragments of SCR1 RNA under glucose-depletion conditions.…”

“…We are interested in a better understanding of the role of Ded1 in the cell. To this end, we used a modified PAR-CLIP technique to identify RNA substrates of Ded1 in vivo [ 24 ]. We identified the Small Cytoplasmic RNA 1 (SCR1) as a major noncoding RNA that crosslinked to Ded1.…”