The RNA helicase Dbp5/DDX19 regulates the ribosomal entry of eRF1-eRF3 and Dom34-Hbs1 in translation termination and cytoplasmic mRNA quality control
by Christian Beißel
Date of Examination:2020-05-18
Date of issue:2020-05-22
Advisor:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Ralf Ficner
Persistent Address:
http://dx.doi.org/10.53846/goediss-7986
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Abstract
English
Translation is a highly regulated and quality-controlled process ensuring the production of correct polypeptides. Although translation initiation is a major target of regulation, translational control occurs also at the end of translation. Both, translation termination and ribosome recycling are tightly connected and create a pool of ribosomal subunits necessary for multiple rounds of translation. Arriving mRNAs from the nucleus are controlled in the cytoplasm for the presence of correct open reading frames (ORFs). In cases where errors are detected, the regarding faulty mRNAs and the truncated proteins are degraded. The three cytoplasmic quality control pathways are nonsense mediated decay (NMD), no-stop decay (NSD) and no-go decay (NGD). NMD targets mRNAs that contain a premature termination codon (PTC), whereas NSD recognizes transcripts that lack a stop codon. NGD finally senses ribosomes that stall on regular codons, because of strong secondary structures or rare codons. In this study, we have characterized the role of the DEAD-box RNA helicase 5 (Dbp5/DDX19) in translation termination. Moreover, we have identified a novel role of Dbp5 in the cytoplasmic mRNA quality control. Current models anticipate that the translation termination factors eRF1 and eRF3 are recruited to terminating ribosomes in a complex. In our studies we used a combination of in vivo and in vitro experiments to shown that Dbp5 regulates a stepwise assembly of the termination complex. Our experiments indicate that the termination factor Rli1 and eRF3-GDP associate with the ribosome first. Subsequently, Dbp5-ATP delivers eRF1 to the stop codon. Dbp5 dissociates upon ATP-hydrolysis, allowing eRF1 to contact eRF3 and terminate translation. Upon GTP hydrolysis by eRF3, eRF1 is placed in the peptidyl transferase center to initiate peptidyl-tRNA hydrolysis. eRF3-GDP is displaced from the termination complex by Hcr1, which was delivered by eIF3. Rli1 can now bind to eRF1 to mediate the release of the peptide. The interaction of Rli1 and eRF1 enables ATP hydrolysis by Rli1, which leads to the splitting of the ribosomes into their subunits. Therefore, the delivery of eRF1 through Dbp5 prevents a premature dissociation of eRF1 from the ribosome through premature contact with eRF3. This is important, because defects in DBP5 result in the readthrough of the stop codon and elongated polypeptides. Thus, the stepwise Dbp5 controlled termination complex assembly is essential for correct translation termination. Defects in DBP5 do not only affect normal translation. Our data furthermore show a function of this helicase in NMD, as an NMD-reporter construct accumulate in RAT8 mutants. Importantly, we have shown that Dbp5 not only collaborates with eRF1 and eRF3 in regular translation and NMD, but we could also show that it regulates NSD through delivery of the non-canonical translation termination factors Dom34 and Hbs1. We show that Dbp5 interacts with Dom34 and Hbs1, which is different from regular termination, where Dbp5 does not contact eRF3. This suggests a different functional mode of Dbp5 in NSD and NGD than in regular termination. In fact, Dom34, and Hbs1 show a decreased interaction with each other and the ribosome when Dbp5 is not functional, supporting this view. Importantly, this binding defect culminates in the accumulation of NSD transcripts. Therefore, Dbp5 does not only govern regular translation termination and NMD via controlling the eRF1 and eRF3 interaction, but it is also an important factor required for NSD and presumable also NGD, which require delivery of Dom34 and Hbs1.
Keywords: Dbp5; translation termination; DDX19; nonsense mediated decay; cytoplasmic mRNA quality control; no-stop decay; no-go decay