Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides

Abstract

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

The translational landscape as regulated by the RNA helicase DDX3

“…While DDX3 affects the global translation landscape, the degree of translation induced varies with individual genes ( 19 , 25 , 33 - 35 ), as revealed by ribosome profiling (also known as ribo-seq) studies ( 36 , 37 ). Indeed, the translation of some mRNAs can be downregulated by DDX3 expression ( 24 , 38 ). In this regard, we highlight recent studies elucidating the role of DDX3 in various types of translation to alter the proteomic landscape in the cell in the presence of diverse intrinsic and extrinsic signals.…”

“…Indeed, a recent study of the DDX3 ortholog of Leishmania infantum showed that loss of DDX3 decelerates ribosome movement along the CDS, causing the emergence of elongation-stalled ribosomes and, consequently, reduced protein production ( Fig. 2B ) ( 38 ). Loss of DDX3 in Leishmania also triggered defective recruitment of the ribosome recycling factor ABCE1 and the termination factors eRF3 and GTPBP1, suggesting inefficient ribosome dissociation and recycling in the absence of DDX3 ( 38 ).…”

“…2B ) ( 38 ). Loss of DDX3 in Leishmania also triggered defective recruitment of the ribosome recycling factor ABCE1 and the termination factors eRF3 and GTPBP1, suggesting inefficient ribosome dissociation and recycling in the absence of DDX3 ( 38 ). Interestingly, prolonged ribosome stalling induced by DDX3 loss leads to ubiquitination of the nascent polypeptide chain with accompanying recruitment of E3 ubiquitin ligases ( Fig.…”

“…Interestingly, prolonged ribosome stalling induced by DDX3 loss leads to ubiquitination of the nascent polypeptide chain with accompanying recruitment of E3 ubiquitin ligases ( Fig. 2B ) ( 38 ). Cotranslational ubiquitination is considered as one of the pervasive proteome quality control mechanisms and is intimately associated with translation ( 80 - 82 ).…”

“…Thus, DDX3 appears to contribute to the maintenance of proteome quality via translation elongation and termination by binding to CDSs and 3’ UTRs of mRNAs ( Fig. 2 ) ( 25 , 38 ).…”