Structural analysis of human UFMylation and ribosome quality control pathways

Penchev, Ivan Rumenov (2025): Structural analysis of human UFMylation and ribosome quality control pathways. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The vast majority of cellular functions are performed by proteins. Protein synthesis, also known as translation, is carried out by the ribosome. It is imperative that translation be as robust as possible, as incorrectly synthesized proteins could potentially lose their function, gain a new function that could have detrimental effects on the cell, or even form toxic aggregates due to misfolding. All of these outcomes can lead to disease in an organism, which is why translation is monitored by a number of pathways to ensure that it is carried out correctly and that any incorrectly synthesized proteins are disposed of. The ribosome quality control (RQC) pathway is one such failsafe. When ribosomes encounter a "roadblock" during translation, translation stalls. Stalled ribosomes not only halt their own translation, but also that of ribosomes trailing behind them, as those collide with the stalled ribosomes. Stalled ribosomes can be split into small 40S and large 60S ribosomal subunits. The large subunit of a split stalled ribosome still carries a transfer RNA (tRNA) bound to an incomplete nascent chain (a peptidyl-tRNA). This 60S-peptidyl-tRNA complex is recognized by the RQC component NEMF, which in turn recruits the E3 ligase LTN1, which ubiquitinates the nascent chain and targets it for degradation. NEMF is also capable of templateless addition of alanines to the nascent chain, also known as CATtailing. Following ubiquitination of the nascent chain, the endonuclease ANKZF1 cleaves the tRNA, releasing the nascent chain from the large subunit, allowing for its degradation by the proteasome. One area of RQC that remains particularly enigmatic is endoplasmic reticulum (ER) specific RQC (ER-RQC). Ribosome stalling at the ER leads to the nascent chain being trapped in the SEC translocon, making it inaccessible for ubiquitination via LTN1. Recent studies on ER-RQC have revealed that the ubiquitin-like modification UFM1 plays an essential role, with UFMylation of the large ribosomal subunit uL24 being necessary for nascent chain degradation. However, the underlying mechanisms remain unclear. The works presented in this thesis focus on ribosome UFMylation and the downstream responses that it elicits. Cryo-EM was used to determine the structure of the trimeric UFM1 E3 ligase (E3UFM1), showing that it has a unique role as both "writer" and "reader" of its own modification. Structural snapshots of the UFMylation reaction combined with release assays confirm that the E3UFM1 itself serves to disassociate 60S subunits from the SEC translocon as a general recycling mechanism. However, the E3 ligase’s initially uncovered binding mode is incompatible with the RQC machinery, as it requires the absence of tRNA, whereas NEMF exclusively recognizes peptidyl-tRNA bound 60S. Structural analysis of UFMylation under stalling conditions uncovered an alternate binding mode of the E3UFM1 component UFL1, in which it can coexist with NEMF on the same 60S, as well as further structural snapshots that showcase the temporal sequence of events in ER-RQC. Taken together, the results showcase UFMylation as a translocon-dissociation mechanism for the large ribosomal subunit, pulling double duty both in traditional termination as well as in ER-RQC via different binding modes of the E3 component UFL1.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 24. Oktober 2025
• 1. Bericht­erstatter:in: Beckmann, Roland