Posttranscriptional regulation of mRNA expression and membrane-associated RNA-binding proteins
We are focusing on how mRNA translation and stability are regulated by RNA-binding proteins and on potential interplays between RNA-binding proteins and endomembranes
1) Mechanisms of post-transcriptional gene regulation.
miRNAs are small non-coding RNAs that have emerged as key regulators of most cellular functions by post-transcriptionally repressing at least 50% of expressed mRNAs. Loaded onto an Argonaute protein, they serve as guide by base-pairing with target mRNAs to recruit a miRNA-induced silencing complex (miRISC) that both represses translation and induces mRNA decay. Decay of targets of miRNAs is well explained by the direct recruitment of the CCR4/NOT deadenylation complex to the miRISC. By contrast, how miRNA induce translation repression is still not fully understood.
Recently we have identified the protein GIGYF2 as a novel miRISC-interacting factor and showed that it specifically promotes miRNA-mediated translation repression (See Fig. 1 and Schopp et al. 2017
). Accordingly, previous studies had shown that GIGYF2 associates with the mRNA 5’-cap-binding protein 4EHP to form a translation inhibition complex (Morita et al. MBC 2012), and a model was put forward in which RBPs recruits the GIGYF2/4EHP dimer to specific mRNAs. We propose that the miRISC acts as such a GIGYF2-recruitment factor.
We recently discovered a second mechanism of GIGYF2-mediated repression that does not depend on 4EHP (See Fig. 1 and Amaya-Ramirez et al. 2018
). In this mechanism GIGYF2 directly binds to its own targets and recruits the CCR4/NOT complex to mediate mRNA decay and translation repression. We have identified a first set of endogenous mRNA targets that are repressed by that mechanism. Interestingly, most of them encode transmembrane or secreted proteins; hence these transcripts are translated by endoplasmic reticulum-associated ribosomes. We are currently analyzing the molecular bases that differentiate the two mechanisms of GIGYF2-mediated repression and their functional outcomes in relevant model cell lines.
Figure 1: a dual mode of GIGYF2-mediated repression depending on whether it is indirectly or directly recruited to its mRNA targets.
2) Interplays between membrane biology and RNA-binding proteins.
Interestingly, the protein GIGYF2 associates with the endoplasmic reticulum (ER) and is thus a membrane-associated RNA-binding protein (Fig. 2). With our finding that ER-associated mRNAs are over-represented among currently identified targets of GIGYF2, this suggests that GIGYF2-mediated repression of mRNAs is an ER-localized process. In addition, work from the Freund lab in Berlin suggests that the GYF domain of GIGYF2 interacts with proteins involved in the COPII pathway (Ash et al., Structure 2010). COPII vesicles are responsible for the first trafficking step of the secretory pathway by transporting all secretory and non-ER resident transmembrane proteins to the Golgi apparatus (See Béthune and Wieland 2018
). We are currently investigating the role of GIGYF2 in the COPII pathway and possible interplays with GIGYF2-mediated repression of mRNAs.
Figure 2: Endogenous GIGYF2 co-localizes with the endoplasmic reticulum
3) Membrane trafficking in polarized cells.
The COPI vesicular pathway mediates important intracellular transport routes by retrieving proteins from the Golgi apparatus to the endoplasmic reticulum, and by transporting proteins within the Golgi apparatus (See Béthune and Wieland 2018
). Interestingly, the COPI pathway was suggested to participate in mRNA localization in neurons (Todd et al., Human Mol Gen 2013). More generally, the exact function of COPI vesicles in highly polarized cells has been hardly studied. We are currently addressing neuron-specific functions of the COPI pathway using a combination CRISPR/Cas9-mediated genome engineering, biochemical, proteomics and microscopy approaches.
4) Tool development for the analysis of protein-protein interactions.
Many of our projects involve the study of protein-protein interaction (PPI) networks. PPIs are often very dynamic and a single protein is typically part of several distinct complexes that remodel according to the exact function that needs to be exerted and to respond to cellular cues. Classical techniques allow identifying all potential PPI a given protein may have but not their functional context. Hence, identifying context-specific functional units is really like finding needles in haystacks. We develop techniques to overcome this inherent challenge of PPI studies by allowing the analysis of context-specific protein complexes. Recently we have expanded the BioID assay, a technique that allows the labeling of proximal proteins within living cells, by creating a split-BioID assay. This technique allows simultaneously discovering or validating binary interactions, and to identify additional proteins that assemble around that specific pair of interacting proteins (See Fig. 3, Schopp et al. 2017
and Schopp et al. 2018
). Visit our split-BioID resources page to know more!
Figure 3: Principle of split-BioID. When proteins A and B interact, the protein fragments NBirA* and CBirA* reassemble an active BirA* enzyme. This leads to the selective biotinylation of proteins belonging to complex I. When the NBirA*-A fusion is part of complex II that does not contain protein B, reassembly of an active BirA* enzyme is not possible. Streptavidin-mediated capture of the resulting biotinylated proteins allows selective proteomics analysis of Complex I.
Download BZH Report Béthune 2014-2016
Proximity-dependent labeling has proved to be a very valuable approach for the study of protein-protein interactions. Its main advantages when compared to co‑immunoprecipitation approaches are that it is less sensitive to transient interactions and that labeling of proximal proteins occurs in intact living cells.
Split-BioID combines the power of a protein-fragments complementation assay (PCA) with the labeling of proximal proteins by the enzyme BirA* used in the BioID approach. Two inactive fragments of BirA* are fused to two putative interacting proteins of interest. Upon interaction of these two proteins, the BirA* fragments are brought in close proximity and re-assemble an active enzyme that mediates the biotinylation of proximal proteins. By carefully choosing the proteins of interest, it allows the analysis of context-specific protein complexes.
If you are interested in trying the approach, the good news is that it is quick and easy!
General split-BioID workflow
Figure from Schopp and Béthune, JoVE 2018
How to perform split-BioID?
If you like action-packed movies with handsome actors, you will find a detailed protocol and demonstration of critical steps here:
All the necessary plasmids are available at Addgene: https://www.addgene.org/Julien_Bethune/
If you cannot afford the handling fees, please contact me.