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Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissue

Pauline E Jullien, Stefan Grob, Antonin Marchais, Nathan Pumplin, Clement Chevalier, Caroline Otto, Gregory Schott, Olivier Voinnet

Preprint posted on December 18, 2018 https://www.biorxiv.org/content/early/2018/12/18/500769

Argonaute 3 facilitates post transcriptional gene silencing in Arabidopsis female reproductive tissues

Selected by Chandra Shekhar Misra

Introduction

The regulation of gene expression is fundamental for the control of cellular homeostasis in plants, animals and fungi. One of the mechanisms of gene regulation is RNA silencing. Silencing of RNA relies on the production of small RNAs which are produced by the cleavage of double stranded RNAs by DICER-like RNAses. These small RNAs are then loaded into the Argonaute (AGO) protein and form RNA induced silencing complexes (RISC). Such complexes promote sequence-specific post-transcriptional gene silencing at the mRNA level or transcriptional gene silencing through changes at the chromatin level (Reviewed in 1 and 2).

In Arabidopsis there are 10 Argonaute proteins, divided into three distinct clades: Argonaute 4, 6, 8, 9, Argonaute 1,5,10 and Argonaute 2, 3, 7. The Argonaute proteins within each clade show a considerable amount of functional redundancy. These proteins perform a diverse range of functions: from plant reproduction to defence against viruses.

Although Argonaute proteins have been extensively studied in plants, several aspects of their function still remain to be elucidated. This preprint describes, for the first time, the role of Arabidopsis Argonaute 3 (AGO3) in plant reproduction and how it differs from Argonaute 2 (AGO2) despite the extensive similarities of both proteins.

Key findings

The authors show that AGO3 is derived from a recent duplication event at the region of AtCOPIA27 transposon in the AGO2 locus.  The corresponding region is rich in arginine and glycine and is therefore called a “glycine-rich repeat” or GRR.

Despite high amino acid sequence conservation between AGO2 and AGO3, no similarity was found between the promoters of the AGO2 and AGO3. This is responsible for their highly distinct, cell type-specific expression pattern: While AGO2 is expressed in both male and female germ cells, AGO3 is expressed in siliques (more specifically within the chalazal integument – a support structure for plant ovules).

Remarkably, differences also extend to the binding capacity of these Argonaute proteins.  AGO3 is able to bind both 21nt and 24 nt sRNAs derived from genic and intergenic regions of the genome. However, AGO2 can only bind 21 nt small RNAs.

In order to better understand the function of AGO3, the authors studied its intracellular localization and found it is mainly localized in the cytoplasm of the stamen filament and of the ovule integument. Interestingly, the mass spectroscopic analysis of AGO3 co-immunoprecipitated proteins revealed that AGO3 was associated with monosome and polysome fractions. This led the authors to propose that AGO3 is involved in post transcriptional gene silencing via its interaction with the translational machinery in siliques.

What I liked about this preprint

Gene expression regulation is a hot research topic. As there are many layers to this process, there is still a lot to understand regarding how cells ensure transcriptional regulation. This matter is particularly interesting in plants, as they have to directly cope with environmental conditions. Not surprisingly, this led to the evolution of different gene regulatory mechanisms during development, growth and reproduction. Although several studies have previously highlighted the role of Argonaute proteins, the mechanisms responsible for gene silencing during reproduction are relatively unknown. This preprint has identified for the first time, how two extremely similar Argonaute proteins (AGO3 and AGO2) have acquired distinct roles in the regulation of plant gene expression in reproduction.

Questions for the authors

The authors have hinted at systemic migration of small RNAs. Thus, it would be really nice to know if AGO3 is involved in small RNAs trafficking in female tissues. If so, it would also be interesting to find out which class of small RNAs (21 nt or 24 nt) would be preferred (AGO3 can bind both).

Since the authors show that AGO3 also possesses cleavage properties, I’m curious to know if they have identified conditions that favour cleavage over translational repression?

The authors put forward the possibility that AGO3 regulates its targets indirectly. Understanding how this is achieved, and to what extent these targets overlap with those of AGO2, would be extremely interesting.

References

  1. Zhang, H., Xia, R., Meyers, B.C. and Walbot, V., 2015. Evolution, functions, and mysteries of plant ARGONAUTE proteins. Current opinion in plant biology27, pp.84-90.
  2. Mallory, A. and Vaucheret, H., 2010. Form, function, and regulation of ARGONAUTE proteins. The Plant Cell22(12), pp.3879-3889.

Tags: arabidopsis, argonaute 3, gene regulation, reproduction

Posted on: 21st January 2019 , updated on: 27th January 2019

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  • Author's response

    Pauline E Jullien shared

    The authors have hinted at systemic migration of small RNAs. Thus, it would be really nice to know if AGO3 is involved in small RNAs trafficking in female tissues. If so, it would also be interesting to find out which class of small RNAs (21 nt or 24 nt) would be preferred (AGO3 can bind both).

    One of our hypotheses is indeed that AGO3 could play a role in long distance sRNA movement as its expression seems restricted to aerial vascular termination. Currently, the classical Arabidopsis hypocotyl grafting technique is limiting to look at such movement toward the female gametophyte as it goes against the source to sink direction. We did try to investigate the effect of the ago3-3 mutation on sRNA systemic movement using this method but we did not see any conclusive effect.

    Since the authors show that AGO3 also possesses cleavage properties, I’m curious to know if they have identified conditions that favour cleavage over translational repression?

    This is a very interesting point but we unfortunately did not identify such conditions. The question of cleavage versus translational repression is still a long outstanding question in the plant AGO field.

    The authors put forward the possibility that AGO3 regulates its targets indirectly. Understanding how this is achieved, and to what extent these targets overlap with those of AGO2, would be extremely interesting.

    As we could not see any overlap between the transcriptome data and the sRNA bound by AGO3, as well as an interaction between AGO3 and the translation machinery, we propose that AGO3 regulate gene expression by translational repression. AGO2 has also been linked, in certain cases, to translational repression. However, in the case of reproductive tissue, as AGO3 and AGO2 expression are not overlapping it is not possible, or would not be relevant, to investigate a potential overlap of targets. It would be very interesting to find conditions, such as stresses or over-expression where both AGO3 and AGO2 could be co-expressed and would allow to investigate this question.

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