Guest Speaker – Julien Bethuné

On Thursday, 30.01.2020, 5.15 pm

Julien Bethuné (Heidelberg University Biochemistry Center), will give an SFB 902 lecture:

„Analysis of context-dependent protein complexes with next-generation proximity proteomics assays“.

Venue: TU Darmstadt, Campus Botanischer Garten, lecture hall: B101/52

Guests are welcome!

Abstract

Since its initial description in 2012, the proximity-dependent biotinylation technique BioID has established itself as a reliable alternative to the classical Affinity Purification-MS approach for the identification of protein-protein interactions. Recently we engineering split-BioID, a proteinfragments complementation assay that greatly enhances the resolution of BioID by allowing the analysis of context-dependent protein complexes. Applied to the miRNA silencing pathway, split-BioID let us identify the 4EHP-binding protein GIGYF2 as a novel miRISC-associated factor. The 4EHPGIGYF2 dimer has been proposed to be recruited by RNA-binding proteins to stimulate the repression of translation of their associated transcripts. In line with this model we found that the
miRISC component TNRC6 directly binds to GIGYF2 which in turns promotes miRNA-mediated translation repression. More recently we uncovered an additional mechanism of GIGYF2-mediated repression that is independent of 4EHP but rather relies on the CCR4-NOT deadenylation complex. This suggests that GIGYF2 is part of two distinct functional units that exert distinct modes of repression on their associated target mRNAs. To define the composition of these context-dependent GIGYF2 complexes, we have developed more versatile proximity labeling assays that rely on a smaller labeling enzyme with improved labeling kinetics and that can be used in a split assay format.

Guest Speaker – Neva Caliskan

On Tuesday, 28.01.2020, 5.15 pm

Neva Caliskan (Helmholtz-Institut für RNA-basierte Infektionsforschung, Würzburg), will give an SFB 902 lecture:

„Single-Molecule and Ensemble Analysis of Protein-mediated Frameshifting“.

Venue: Campus Riedberg, lecture hall: N260/3.13

Guests are welcome!

Abstract

Three bases encoding for an amino acid seem to represent the universal feature of the genetic code, yet ribosomes have evolved to read the code in different ways by altering the triplet periodicity of the reading frame. This phenomenon is called programmed ribosome frameshifting (PRF). PRF requires specific cis-acting elements – a slippery site followed by a stable RNA structure. PRF efficiency is also affected by trans-acting factors, including proteins, miRNAs and metabolites. While the general mechanisms of PRF and the involvement of cis-acting elements in this process are well understood, the regulation of these events is still vastly understudied. Additionally, the interactions of these factors with the RNA and the translation machinery have not yet been completely understood. Recent advances in single-molecule techniques allow to study these events at the molecular level and thus unveil hitherto unrecognised details. In this study, we chose the encephalomyocarditis virus (EMCV) 2A protein as a model to study PRF regulation. The expression of this protein is essential for frameshifting on the EMCV mRNA, and inhibition of PRF leads to severely reduced virulence. We investigated the interplay of the 2A protein with its frameshifting-RNA target. To do so, we combined single-molecule techniques, such as optical tweezers and confocal microscopy, together with HPLC-MS and microscale thermophoresis (MST). We anticipate these assays to be a starting point in analysing the translational kinetics of frameshifting and its interplay by RNA binding factors. Furthermore, recent examples of identification of such factors indicate that they play a major role in PRF regulation and understanding their mode of action will certainly uncover new fundamental principles of RNA-based gene regulation.

Guest Speaker – Boris Görke

On Thursday, 12.12.2019, 5.15 pm

Boris Görke (Max-Perutz-Labs, Wien), will give an SFB 902 lecture:

„A regulatory network controlling bacterial cell envelope synthesis at the post-transcriptional level“.

Venue: TU Darmstadt, Campus Botanischer Garten, lecture hall: B101/52

Guests are welcome!

Abstract

Gram-negative bacteria such as E. coli possess a cell envelope consisting of peptidoglycan and an outer membrane, which provides protection against environmental stresses. Synthesis of glucosamine-6-phosphate (GlcN6P) by enzyme GlmS represents the first and rate-limiting step in the cell envelope synthesis pathway. In order to ensure continuous cell envelope synthesis, GlcN6P homeostasis is required, which is achieved through a post-transcriptional regulatory circuit composed of small RNAs (sRNAs) GlmY and GlmZ and the RNA-binding protein RapZ. GlmZ activates glmS translation by base-pairing. When GlcN6P is ample, GlmZ is bound by adaptor protein RapZ and inactivated by cleavage through recruitment of RNase E. Decreasing GlcN6P concentrations provoke accumulation of the homologous sRNA GlmY, which acts as decoy and sequesters RapZ thereby counteracting GlmZ decay. The crystal structure of RapZ revealed a novel type of RNA-binding protein, which putatively evolved from re-purpose of a glycolytic enzyme. The active form of RapZ corresponds to a tetramer, which may activate the likewise tetrameric RNase E in an encounter complex to cleave sRNA GlmZ that is presumably sandwiched between the two tetramers. Notably, RapZ also represents the sought GlcN6P sensor: RapZ binds GlcN6P with high affinity and rapZ mutants are blind for this metabolite. Upon GlcN6P depletion, RapZ stimulates a transcription factor through interaction to increase GlmY expression levels. Thus, RapZ has a moonlighting function and controls production of its own decoy GlmY, which subsequently sequesters the available RapZ protein into long-lived ribonucleoprotein complexes, thereby counteracting GlmZ decay.