Nuclear siRNA in chromatin dynamics – Nuclear siRNAs

We are using the recent technics of in vivo recombineering to manipulate genes involved in small RNA biogenesis and activity in order to introduce flag epitopes in their sequence. It is then possible visualize the corresponding proteins in microscopy and to analyze their nucleo-cytoplasmic distribution using appropriate anti-flag antibodies.
Next Generation Sequencing (NGS) Technologies allow to sequence small RNAs, to map them in the genome, to evaluate their expression levels, and to identify potential structural or sequence modifications. When associated to genetic approaches, NGS also allows to map the nuclear proteins involved in small RNA biogenesis or activity.
Bioinformatic approaches are required to mine NGS datasets. We are developing these approaches using the Galaxy framework, which allows integration of complex algorithms in an open web platform accessible to biologists and medical doctors

- Paramutation in Drosophila linked to emergence of a piRNA-producing locus
- Convergent evolution of argonaute-2 slicer antagonism in two distinct insect RNA viruses
- A Galaxy server dedicated to bioinformatic analyses of small RNA sequencing datasets is now available on line through a WEB interface

Our work provides a model for analyzing the emergence of piRNA loci and to better understand inherited epigenetic transitions based of small RNA transmission
Notre travail sur la paramutation fournit un modèle précieux pour l'analyse mécanistique de
We also wish to study the long-term effects, over generations, of a high viral siRNA load in organisms.
We are considering with the University Paris 6 and the CNRS the creation of a spin-off company dedicated to the identification of new biomarkers and therapeutic targets in the small RNA pathways

1. Jouneau et al. (2012). Rna 18, 253-264. The work shows that naive and primed murine pluripotent stem cells have distinct miRNA expression profiles.
2. van Mierlo, J.T. et al (2012). PLoS Pathog 8, e1002872. The work shows convergent evolution of argonaute-2 slicer antagonism in two distinct insect RNA viruses.
3. de Vanssay, A. et al (2012). Nature. The work identified a paramutation in Drosophila linked to emergence of a piRNA-producing locus
4. Antoniewski, C. (2011). Methods in molecular biology 721, 123-142. In this work, I describe Visitor, an informatic pipeline for analysis of viral siRNA sequencing dataset

20-30nt small RNAs are at the core of the RNA silencing biochemical framework. They associate with Argonaute family proteins, which they guide to complementary targets. The resulting silencing occurs by cleavage, destabilization or inhibition of translation of RNA transcripts (Post Transcriptional Gene Silencing), or by DNA or histone modifications at complementary loci (Transcriptional Gene Silencing). Small RNAs are involved in numerous biological processes including gene regulation, defense against exogenous pathogens as well as endogenous selfish transposons, chromatin and chromosome dynamics and genome rearrangements. This variety of mechanistic and functional outputs directly reflects the multiplicity of recently uncovered RNA silencing pathways.

The role of small interfering RNAs (siRNAs) in chromatin structure regulation has been characterized in detail in plants and in the unicellular eukaryote Schizosaccharomyces pombe. In contrast, the contribution of small RNA pathways to chromatin dynamics in animals remains poorly understood. Yet, deciphering the mechanisms that control chromatin structure is a challenge of first importance in biology, as there is a wealth of evidence that their misregulation is involved in genome instability, (epi)genetic diseases, cancers and aging.
We recently demonstrated in Drosophila melanogaster that sequestering Transposable Element derived siRNAs in the nucleus alters histone H3-K9 methylation and chromosomal distribution of the histone methyl transferase Su(var)3-9 and of the Heterochromatin Protein 1, and suppresses silencing of heterochromatin gene markers. The same effects were also observed in dcr2, r2d2 and ago2 mutants. Hence, our results provided evidence of a nuclear siRNA pathway whose function is to facilitate heterochromatin formation in Drosophila somatic cells.

The goal of the proposed project is to explore this newly uncovered small RNA silencing pathway and to characterize its role in heterochromatin dynamics.
Our objectives are:
(i) to purify nuclear siRNA, to characterize their sequences, structures and genome locations and to test the role of heterochromatin components such as Su(Var)3.9 and Heterochromatin Protein 1 in their biogenesis.
(ii) to take advantage of the most recent tools in Drosophila functional genomics (BAC recombineering coupled to attP targeted transgenesis) to generate a resource of genomic transgenes expressing the components of the siRNA pathway (Dcr2, R2D2, Ago2, etc…) tagged with FLAG-HA and GFP epitopes.
(iii) to use this resource to characterize in detail the nuclear localization of Dcr2 and Ago2 proteins and define their interactions with other nuclear components through in vivo and in vitro imaging as well as in biochemical approaches.
(iv) To map Dcr2 and Ago2 chromosome binding sites at genome wide level using chromosome immunostaining and ChIP-seq approaches.

This study should ultimately allow a better understanding of the processes involved in chromatin structure regulation and epigenetic cellular heredity and facilitate their exploitation for improvement of health.