Can histone code-like switches govern the multifunctionality of RNA-binding proteins?

Informations projet

MARS

N° de convention de subvention: 753803

DOI

10.3030/753803

Projet clôturé

Date de signature de la CE 24 Mars 2017

Date de début 1 Avril 2017

Date de fin 31 Mars 2019

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 183 454,80

Contribution de l’UE

€ 183 454,80

Coordonné par

THE UNIVERSITY OF EDINBURGH
United Kingdom

Periodic Reporting for period 1 - MARS (Can histone code-like switches govern the multifunctionality of RNA-binding proteins?)

Période du rapport: 2017-04-01 au 2019-03-31

Résumé du contexte et des objectifs généraux du projet

DNA contains the information for making the proteins that our cells need to function normally. This process is known as “gene expression” and our health relies on it being tightly controlled to ensure that the right proteins are made in the right cells, time, and amount.
Gene expression is a two-step process where a transient intermediate called messenger RNA (mRNA) is copied from the DNA and used as a template for making proteins. We aimed at understanding how mRNA fate is controlled. We focus on a class of proteins: “RNA-binding proteins” (RBPs) that can attach to mRNAs, to act as regulators. However, a major gap in our knowledge is how their different functions are controlled such that the correct proteins are synthesised.
We focused on one well-studied multifunctional RBP called poly(A)-binding protein 1 (PABP1), which is a critical regulator of mRNAs. Previous studies established that PABP1 interacts with multiple proteins and that those different partners are involved in PABP1 different functions. However, many partners interact with the same piece of PABP1, making it difficult to understand how it interacts with the correct partner, at the right time. Our previous work had revealed a potential clue as we found that PABP1 is subject to many post-translational modifications (PTMs). Proteins are chains of amino acids (AAs) and PTMs are chemical groups that are added to specific AAs to change their properties. This can for instance block interaction with protein partners. In particular we found that one AA that is key for many partner interactions is subject to more than one type of mutually exclusive PTM. Therefore, we hypothesised that this may provide a “switch” that determines which protein partner interacts with PABP1, hence dictating its function.
Since these types of PTMs have recently been identified in the >1000 human RBPs, our research could provide an important paradigm for how different functions of multifunctional RBPs are co-ordinated to achieve finely tuned gene expression.
Information gained has practical applications. RBPs are important as their dysfunction causes many type of diseases including metabolic, reproductive, neurological and oncogenic disorders. Understanding how they function is a necessary step to understand how they go wrong in disease, and to develop therapeutic compounds.
Knowledge gained can also be used by commercial sectors which rely on biology: Analysis of the EU “bioeconomy” in previous years of has shown it to account for €2.1 trillion annual turnover and 18.3 million jobs.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

We applied a combination of established and cutting-edge technologies to understand how mutually exclusive PTMs at a single AA in PABP1 control its interactions with protein partners. Use of a novel “synthetic biology” state of the art approach was key to addressing this question and a manuscript reporting the developed/optimised method will be submitted shortly. Synthetic biology is an approach where cells are “engineered” into factories to make specific proteins. This allowed us to generate PABP1 and control the PTM status of specific AAs. Other key tools were also developed for this project, including antibodies that differentiate between PABP1 with different PTM status. These antibodies will be made available to other researchers following publication.
Using this approach, we generated three different versions of PABP1 and were able to test their interaction with domains from 16 protein partners. Remarkably these partners all interact with the same area of PABP1 but are involved in very different functions. Excitingly, while the strength of interaction of PABP1 for most partners was unaffected, two critical partners showed increased affinity for PABP1 with specific PTMs.
To understand this new layer of gene expression regulation, we used a structural biology tool: X-ray crystallography. This technique explores the 3-dimensional shape of proteins, down to the individual atoms and can show how they interlock with other proteins or compounds (e.g. drugs). Our experiments revealed how one specific PTM alters the “surface” of the protein. We also studied how the docking of proteins on PABP1 was affected by the PTM. Unexpectedly, we discovered that the PTM diverts the “track” where the partners usually lie, allowing multiple stabilizing interactions, explaining the enhanced affinity. This represents the first evidence that RBPs can utilise these particular types of PTM “switches” to coordinate partner binding. A manuscript reporting these exciting findings will shortly be submitted for publication.
To investigate how these changes impact living cells, we investigated PABP1 PTM status in cells in culture. Using our unique PABP1 antibodies, we revealed that cells respond to specific environmental cues by changing PABP1 PTM status. Cells respond to their environment by changing gene expression, and we can predict which aspects of PABP1 function are changed from our in vitro study. Current work is investigating this final piece in the puzzle and we have developped a series of tools. These include “forcing” human cells to express PABP1 proteins with specific AA substitutions that “mimic” the PTMs, a series of “reporters” that allow different aspect of PABP1 function to be assessed, and we are combining this with a cutting-edge system to remove the PABP that is normally present in cells such that only our PABP1 variants are present. This final work will be presented at an international conference in July 2019.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

This project has potential impacts on basic academic research or applied research including biomedical, biotech, pharma and industry.
It represent a first insight into the effect of PTMs on mammalian PABPs. It will also provide an important paradigm establishing that this type of PTM switches can control RBP multifunctionality, a mechanism only described to regulate DNA-binding proteins.
Our cutting-edge synthetic biology approach establishes a precedent for the study of other RBPs in gene expression regulation, (>1000 in human cells) an increasing number of which are being identified as involved in human pathologies.
Our results may also lead to new therapeutic strategies, as PABP1 is the target of many pathogenic viruses, and is present in pathological deposits in a number of neurodegenerative diseases. Moreover, its functions include the correct termination of protein synthesis, which is aberrant in about 20% of clinical conditions caused by point mutations, thus our results could help improve on drugs that recently entered clinical use that target this process.
“Synthetic biology” is an important area for the future where cells of different species are “engineered” to make specific types of proteins for industrial or therapeutic use e.g. food flavourings, biofuels or novel antibiotics. It is also useful for bio-economy including traditional industries such as brewing where manipulating gene expression can improve yeast strains for the fermentation as well as the resultant taste profiles.
As RBPs are key regulators of gene expression, understanding their regulation offers an opportunity to modulate their function for large-scale synthesis of particular proteins.
To achieve these impacts, work has been presented at six meetings including local and international events, and will be published in journals and tools generated will be made available to other researchers.

Fig 1.Proteins containing a PABP-interacting motif "PAM2" confer PAB multifunctionality.