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Schmidt, Christian (2017): A physical link between mRNA translation and degradation: structures of the Ski complex and eIF-5A bound to the ribosome. Dissertation, LMU München: Fakultät für Chemie und Pharmazie
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Abstract

mRNA translation and degradation are mutually interdependent processes in the cell. The best characterized examples of the interplay between these two processes are the mRNA quality control pathways taking care of aberrant mRNAs that cause translation stalling in most cases. It was shown for these pathways that mRNA degradation is initiated in a ribosome-dependent manner directly on the stalled intermediate. Aberrant transcripts are either degraded in 5’-to-3’ direction by Xrn1 or in 3’-to-5’ direction by the cytosolic exosome together with the Ski proteins. However, no structural insights exist on how translation and degradation are coupled for general mRNA turnover and quality control. Beside ribosome stalling on aberrant transcripts, poly-basic or poly-proline stretches have been shown to cause translation arrests in the cell. Yet, these stretches are commonly found in proteins. To that end, eukaryotic initiation factor 5A (eIF-5A) was identified to rescue ribosomes stalled on poly-proline, allowing translation to continue. Moreover, eIF-5A was shown to alleviate many poly-basic stalling events and promoting translation elongation in general. It is, however, unknown how eIF-5A functions on a molecular level. The first part of this thesis focuses on the interactions of the Ski proteins with ribosomes in the exosome-dependent 3’-to-5’ mRNA degradation pathway. We show that in contrast to most proposed models, the Ski complex and not Ski7 associates stably with ribosomes in vitro and in vivo. A high resolution cryo-EM structure of a native ribosome-Ski complex reveals how the Ski complex interacts with the 40S subunit of the ribosome, facilitating the threading of mRNA into the Ski2 helicase. Furthermore, we show by ribosomal profiling analysis that this interaction is probably not limited to mRNA quality control, but might rather represent a general mRNA turnover intermediate. Collectively, these results are the first structural insights into how translation and degradation of mRNAs are coupled on a molecular level. The second part of this thesis focuses on the surprising discovery that eIF-5A binds to Ski complex-associated ribosomes. We show that eIF-5A targets ribosomes with a vacant E-site, thus recognizing translation-arrested intermediates by scanning for tRNA occupancy. A cryo-EM structure reveals that eIF-5A reaches deep into the peptidyl transferase center and interacts with A76 of the P-site tRNA via its unique hypusine residue. Our structural data supports a model where this interaction leads to the stabilization and orientation of the P-tRNA CCA-end to assist in peptide-bond formation, explaining eIF-5A’s function as a general rescuing factor.