Analysis of eukaryotic translation by integrating cryo-EM and ribosome profiling

Shanmuganathan, Vivekanandan (2023): Analysis of eukaryotic translation by integrating cryo-EM and ribosome profiling. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Translational control plays a critical role in maintaining proteome homeostasis, and in influencing cellular differentiation, proliferation, growth and developmental pathways. Protein synthesis is closely linked to cellular metabolism and any aberrations in its regulation lead to diseased states. In this thesis presented here, translational regulation has been investigated in three different biological contexts by integrating two different techniques namely, cryo-electron microscopy and ribosomal profiling. Translational regulation has been investigated in maturing dendritic cells using ribosome profiling and RNAseq respectively. Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In the immature state (immature DC), they have the ability to monitor the environment and upon encountering antigens they mature to launch immune responses. Here, a defined cytokine mixture combined with TLR agonist (R848) has been used for in vitro DC maturation. Upon induction of maturation, pathways such as the ‘TNF signaling pathway’, the ‘cytokine-cytokine receptor interaction’ and the ‘IL-17 signaling pathway’ were up regulated both at the level of transcriptome and translatome respectively. Transcripts encoding for proteins involved in oxidative phosphorylation pathway were strongly repressed at the later stages of DC maturation (24 h). As observed in previous studies transcripts encoding for ribosomal proteins, antigen processing and presentation were also translationally up-regulated at 4 h while being translationally repressed at the 24 h time point. Transcripts of the glycolytic pathway are also translationally repressed at the 24 h time-point. Further, during the course of DC maturation, globally there was increased ribosome occupancy in the 5’ UTR. During the later stages of DC maturation, down regulation of ABCE1 led to accumulation of post-termination ribosomes in the 3’ UTR. Moreover, ribosome occupancy in the 3’ UTR showed strong correlation to its GC content. Ski proteins function as accessory factors and are essential for exosome function, which mediates the 3’ to 5’ mRNA decay pathway. Non-stop transcripts are primarily decayed via the 3’ to 5’ pathway. It has been shown here that the Ski complex, interacts with the ribosome independent of Ski7. Ribosomal profiling of 80S-Ski-complexes revealed a fraction of longer footprints, and contained more poly-A containing footprints. Further, RNAseq analysis of the purified 80S-Ski-complexes revealed strong asymmetric distribution of reads, where more reads mapped towards the 5’ end of the transcripts. Also, transcripts with shorter half-life (< 5 min) and with more non-optimal codon content showed enrichment for Ski-80S footprints. This hinted at the possibility that Ski complex might interact with ribosomes for turnover of canonical transcripts via the 3’-5’ decay pathway. The endoplasmic reticulum (ER) is responsible for properly modifying and folding most of the secretory and membrane proteins. Its functioning capacity is challenged during stressful circumstances such as in hypoxia, calcium imbalance and viral infection. Unfolded protein response (UPR) is the cellular mechanism that is activated to alleviate the ER stress. UPR acts via three main pathways in mammals, and of this IRE1α-XBP1u branch is the most evolutionarily conserved. XBP1u contains a C-terminal ribosomal pausing site and plays a critical role in mediating UPR. Using cryo-EM, XBP1u has been visualized in the ribosomal exit tunnel. Structural characterization revealed that XBP1u forms a turn in the vicinity of the peptidyl transferase center and causes a subtle distortion of the base C4398 to inhibit ribosomal activity. This explains the temporary nature of the ribosomal arrest mediated by XBP1u. During ribosomal pausing, HR2 of XBP1u is being recognized by SRP, but it fails to successfully engage with the Sec61 translocon. XBP1u has evolved with an intermediate ribosomal pausing strength, but this allows it to be efficiently targeted by SRP onto the Sec61 translocon, albeit without gating it.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Ribosome, Eukaryotic translation, Unfolded protein response, Ribosome profling
• 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: 3. März 2023
• 1. Bericht­erstatter:in: Beckmann, Roland