PASRNA

Deciphering post-translational control of Argonautes and their effects on small RNA homeostasis (PASRNA)

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 Obiettivo del progetto (Objective)

'Argonaute (AGO) proteins are integral components of most small RNA silencing pathways in plants and metazoans. Previous studies in both model systems have demonstrated an inherent link between AGO levels and the steady state expression of global populations of miRNAs, strongly suggesting a layer of homeostatic control. In an effort to understand this phenomenon, we have set out on, a series of experimental approaches to uncover and characterise the mechanisms underlying AGO post-transcriptional control. Under normal physiological conditions, and in response to viral infection, we will monitor how the turnover and activity of AGO impacts the global small RNA equilibrium in both plants and metazoans. We have identified a potentially conserved mechanism, whereby Cullin RING-E3 ubiquitin Ligases (CRL) negatively regulate AGOs functioning in miRNA-mediated gene repression. Employing both plant and mammalian models systems in a multidisciplinary approach, we aim to dissect the role of CRLs’ activity on small RNA pathways and AGO1 biology, using a range of biochemical, genetic and molecular biology techniques. In addition, we will employ a proteomic-based screen to identify the full degree of AGOs post-translational modifications. Collectively these approaches should unravel novel modes of AGO regulation through the control of subcellular localisation, protein-protein interactions or RISC assembly/recycling. Overall, the proposed systematic investigation of the currently elusive mechanism(s) underlying AGO regulation and small RNA homeostasis will provide greater insights into fundamental aspects of small RNA biology. This is pivotal to further our knowledge regarding the therapeutic, veterinary and agronomical exploitation of these molecules.'

Introduzione (Teaser)

Researchers have taken steps towards better understanding a control mechanism present in plant and animal cells.

Descrizione progetto (Article)

RNA interference (RNAi) is a complex system of cellular regulation that researchers are still trying to fully comprehend. Recent evidence suggests that Argonaute (AGO) proteins control RNAi in the cell, but the mechanisms of this control are not very clear.

The EU-funded PASRNA project investigated one possible mechanism: post-translational modifications (PTMs) of the AGO proteins.

PTMs are changes that happen to a protein after it is produced by the cell, usually as a way to 'switch on' or 'switch off' the activity of that protein. PASRNA investigated the phosphorylation and ubiquitination of AGO, two of the most common PTMs.

Researchers managed to identify a specific location on the AGO protein where phosphorylation takes place. It is in the functional core of the protein where it binds to various types of RNA, suggesting that phosphorylation affects AGO function.

PASRNA then tested this in human immune cells, where RNAi plays an important role. When these cells were exposed to pathogens, AGO was phosphorylated rapidly (within an hour) and then phosphorylation slowly decreased over time.

An investigation into ubiquitination of AGO was less conclusive. Researchers did however find an enzyme that is responsible for ubiquitinating AGO.

Overall, the research of PASRNA has confirmed that AGO control of RNAi occurs through PTMs of the AGO protein in response to external stimuli. This improves our knowledge of RNA biology and will have knock-on effects for human, animal and plant health.