Investigating the functional role of mammalian ribosomal protein uL22

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Abstract

The ribosome is a ribonucleoprotein complex which translates mRNA into protein. During translation the nascent polypeptide chain passes through a confined exit tunnel environment which can allow protein folding to occur. As the nascent-chain emerges from the exit tunnel and enters a crowded cellular environment which is aggregation prone, a variety of chaperones, enzymes and targeting factors compete for the emerging substrates in a regulated manner. Thus, the fate of proteins may need to be decided early on during its synthesis. Recent evidence has emerged that the ribosomal exit tunnel has a more functional role than previously thought. Peptide sequences within the nascent-chain can signal to the peptidyl transferase centre (PTC) to induce translational stalling from within the exit tunnel. In addition, there is a potential role of the exit tunnel for the recruitment of protein biogenesis factors (PBFs). Essential to the exit tunnel’s functions is the ribosomal protein uL22. uL22 is exposed at the surface of the ribosome but possess a β-hairpin that extends into the ribosomal exit tunnel and its tip forms a constriction in the tunnel which can interact with the nascent-chain. Mutations in this loop can abolish translational stalling in bacteria. Also, the loop can interact with a transmembrane segment in a manner which has been proposed to modulate the ribosome exit site that PBFs can bind to. Despite uL22 being conserved, the role mammalian uL22 plays is poorly understood. The mammalian peptide XBP1u induces stalling to ensure efficient splicing of its own mRNA during the unfolded protein response (UPR), which is involved in resolving ER stress. UPR is important as unresolved ER stress can lead to diseases such as diabetes. However, the mechanism in which XBP1u induces stalling from within the mammalian ribosomal exit tunnel remains to be investigated. Thus, the aim of the thesis was to investigate the functional role mammalian uL22 plays in the ribosomal exit tunnel. This was to provide insight on how the mammalian ribosome is able to process information on the nature of the nascent-chain in order to influence translation or affect its fate along a crowded cellular environment. In this thesis, mammalian uL22 was shown to be able to functionally replace yeast uL22 indicating a degree of functional conservation between the two species. A system was then developed to assess the function of mammalian uL22 in mammalian cells by making mutations in the conserved β-hairpin loop of uL22. Specific mutations affect XBP1u stalling and in a manner that affects efficient splicing of XBP1u mRNA during ER stress. Mutations of uL22 can also affect the recruitment of the ER-targeting factor SRP to translating ribosomes possibly in a manner that affects levels of newly-made glycosylated proteins in the ER. Overall, this study provides first evidence of the key role mammalian uL22 plays within the exit tunnel in regulating translation, as in the case of XBP1u to ensure efficient UPR, and can influence the recruitment of SRP to affect the fate of the emerging nascent-chain. Further investigation is needed in order to get a more direct mechanism of how the mammalian uL22 β-hairpin is able to communicate to the PTC, in the case of XBP1u, and the ribosome exit site to influence PBF recruitment.

Keyword(s)

Ribosome; Translation regulation; Unfolded protein response

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