Molecular recognition of aberrant translation

Protein translation is a fundamental, demanding process which requires several million ribosomes and consumes as much as 75% of cellular energy. Because of its importance, translation is regulated at many levels to maintain its high fidelity, with both substrates and synthesized products monitored by numerous quality control mechanisms. While post-translational quality control of proteins has been studied extensively, the mechanisms of co-translational quality control of nascent polypeptides, mRNA and the ribosome itself have only recently been appreciated. One of the major unanswered questions is how quality control mechanisms manage to specifically identify an aberrant event amid widely heterogenous normal physiologic states. This question forms the basis of this thesis, which is focused on understanding the molecular principles that determine accurate recognition of aberrantly slow ribosomes. To address this, we developed a novel flow cytometry-based assay to visualize terminal ribosome stalling at single cell resolution in mammalian cells. Using the assay, we firmly established that poly(A) messenger RNA (mRNA) is the most potent cause inducing terminal stalling. This system also led us to the identification and downstream characterization of a novel protein factor, the E3 ubiquitin ligase ZNF598, which we showed to be involved in triggering the quality control pathway during poly(A) translation. Subsequent in vitro ubiquitination experiments using purified ribosomes and ligase revealed molecular targets of ZNF598 - proteins eS10 and uS10 of the 40S ribosomal subunit. We further verified that ubiquitination of both targets is functionally important for poly(A)-mediated terminal stalling in cultured cells. Together, it led to the conclusion that ZNF598 recognizes excessively slow ribosomes and ubiquitinates them on the small subunit to initiate downstream quality control pathways responsible for the degradation of aberrant nascent proteins. To further understand how ZNF598 specifically recognizes and ubiquitinates aberrantly translating ribosomes, we reconstituted its recruitment to poly(A)-stalled translation complexes in an in vitro translation system. Unexpectedly, these experiments revealed that ZNF598 specifically associates with and ubiquitinates a sub-population of poly(A) stalled ribosomes consisting of closely-packed, collided di-ribosome species. This finding led to the general model of indirect detection of excessively slow ribosomes by ZNF598, whereby it recognizes ribosome collision events. We subsequently verified and generalised our proposed model using in vivo based experiments. In cultured cells, induction of stochastic ribosome collisions using sub-inhibitory doses of several unrelated translation elongation inhibitors led to the robust recruitment of ZNF598 to the sites of collisions, as manifested by ubiquitination of eS10. Our results explain the mechanism for sensing excessively slow translation at the molecular level. Moreover, the proposed model has also profound implications for general cellular physiology. Most importantly, the use of ribosome collisions to infer stalling means the degree of slowdown tolerated on an mRNA is tuned by the frequency of translation initiation; hence, the threshold for triggering quality control is necessarily substrate-specific

Ribosome collisions trigger cis-acting feedback inhibition of translation initiation.

Translation of aberrant mRNAs can cause ribosomes to stall, leading to collisions with trailing ribosomes. Collided ribosomes are specifically recognised by ZNF598 to initiate protein and mRNA quality control pathways. Here we found using quantitative proteomics of collided ribosomes that EDF1 is a ZNF598-independent sensor of ribosome collisions. EDF1 stabilises GIGYF2 at collisions to inhibit translation initiation in cis via 4EHP. The GIGYF2 axis acts independently of the ZNF598 axis, but each pathway's output is more pronounced without the other. We propose that the widely conserved and highly abundant EDF1 monitors the transcriptome for excessive ribosome density, then triggers a GIGYF2-mediated response to locally and temporarily reduce ribosome loading. Only when collisions persist is translation abandoned to initiate ZNF598-dependent quality control. This tiered response to ribosome collisions would allow cells to dynamically tune translation rates while ensuring fidelity of the resulting protein products

The architecture of EMC reveals a path for membrane protein insertion.

Funder: Boehringer Ingelheim Fonds; FundRef: http://dx.doi.org/10.13039/501100001645Funder: Naito Foundation; FundRef: http://dx.doi.org/10.13039/100007428Funder: Japanese Biochemical SocietyApproximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function

The architecture of EMC reveals a path for membrane protein insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function

The architecture of EMC reveals a path for membrane protein insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function

The architecture of EMC reveals a path for membrane protein insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function

Application of SELEX technique in selection of RNA aptamers targeting murine VCAM-1

Aptamery to krótkie, jednoniciowe oligonukleotydy DNA lub RNA zdolne do wysoce specyficznego rozpoznawania i wiązania różnych celów molekularnych. Uzyskuje się je w procesie selekcji in vitro zwanym SELEX (ang. Systematic Evolution of Ligands by EXponential enrichment), polegającym na wybiórczej identyfikacji i amplifikacji aptamerów wiążących cel spośród cząsteczek biblioteki kombinatorycznej zawierającej do 10^15 różnych aptamerów. Dzięki swoim niezwykłym właściwościom aptamery posiadają bardzo szerokie zastosowanie m.in. w przemyśle medycznym służąc zarówno jako narzędzia diagnostyczne jak i terapeutyki.Celem pracy było uzyskanie modyfikowanych aptamerów RNA wiążących białko VCAM-1 wykorzystując metodę SELEX. Białko VCAM-1, to cząsteczka adhezyjna z nadrodziny immunoglobulin, której główną funkcją jest pośredniczenie w aktywnej migracji leukocytów przez ścianę śródbłonka naczyń do miejsc występowania stanu zapalnego. Podwyższony poziom tego białka zarówno w formie błonowej, jak i rozpuszczalnej koreluje z wieloma stanami patologicznymi m.in. miażdżycą naczyń, co czyni je dobrym markerem molekularnym chorób śródbłonka. Aptamery wiążące VCAM-1 mogą być wykorzystane jako potencjalne narzędzie do diagnostyki dysfunkcji śródbłonka zarówno in vivo jak i ex vivo.Aptamers are short, single-stranded DNA or RNA oligonucleotides, which can specifically recognize and bind the whole range of molecular targets. Aptamers can be generated in a process of in vitro selection called SELEX (Systematic Evolution of Ligands by EXponential enrichment). The principle of SELEX is based on a selective identification and amplification of aptamers specific against a target molecule from the combinatorial library of up to 10^15 different molecules. Aptamers, because of their unique features, have a broad spectrum of applications, amongst others in the field of biomedicine where they are being used as diagnostic tools as well as therapeutics. The aim of this work was to obtain modified RNA aptamers targeting VCAM-1 using SELEX technique. VCAM-1 is an adhesion molecule from the immunoglobulin superfamily, whose main function is related to the process of leukocyte migration through the endothelium to the sites of inflammation. Increased levels of both forms of this protein, transmembrane as well as soluble, correlates with many pathological states including atherosclerosis, which makes it a good biomarker of vascular diseases. Therefore, aptamers that selectively bind VCAM-1 can be potentially used as a diagnostic tool in detection of endothelial dysfunction in vivo as well as ex vivo