Over the last decade, small, non-coding RNAs emerged as core components of a vast silencing framework in animals as in plants. They fulfill critical roles as transcriptional and post-transcriptional regulators and as guides of chromatin-modifying complexes; they are involved in numerous biological processes including development, defense against transposons and pathogens, and chromosome dynamics. This fascinating variety of mechanisms and functional outputs reflects the multiplicity of intertwined RNA silencing pathways that interact with each other. Our goal is to contribute at understanding the molecular basis of these variations and their biological impact.
Three types of small RNAs, piRNAs, miRNAs and siRNAs, accumulate in different tissues and developmental stages of Drosophila. The core “panoply” involved in their biogenesis and activity includes 3 RNase III enzymes, Drosha, Dicer-1 (Dcr-1) and Dicer-2 (Dcr-2) and 5 Argonaute proteins, Ago-1, Ago-2, and the three germinal Argonautes Piwi, Aubergine (Aub) and Ago3.
- The 25-27nt piRNAs are produced in the gonads from a limited number of pericentromeric or telomeric loci that are composed of nested repeats of ancient transposable elements. They are loaded in the germinal Argonautes Piwi, Aub and Ago3 and guide the cleavage of transposon transcripts. piRNAs can be envisioned as sentinels for transposon invaders: when newly invading Drosophila transposons “fall” into piRNA producer loci, they become templates for the production of complementary piRNAs, which trigger transposon silencing in a negative feedback regulatory loop.
- The ~22 nt miRNAs derive from structured precursor transcripts called primary miRNAs (pri-miRNAs) whose nuclear processing by the RNase III Drosha associated to its partner Pasha gives rise to pre-miRNAs. Pre-miRNAs have a typical ~70nt hairpin structure; the stem of the hairpin always includes mismatches that result in “bulges”. They are exported to the cytoplasm where Dicer-1 associated with its partner Loqs cuts out the loop and liberates a 22nt miR/miR* imperfect duplex. The miR strands are mainly loaded into Ago-1 and guide translational repression as well as destabilization of target mRNAs whose 3’ UTR contains target motives complementary to the most 5’ 7-8 nt of the miR sequence, called the seed sequence. In fact, finding relevant miR target sites in mRNAs and establishing their true regulatory role remains one of the most difficult challenges in miRNA biology. The picture is further blurred by the presence of combinations of putative seed targets for different miRNAs in a given 3’ UTR, as well as by the ability of a given miRNA to target several hundred different mRNAs. miRs*, also called “passenger strands” were initially considered a “by pass” product of miRNA biogenesis. Increasing evidence indicates that they are indeed loaded into Ago1 in a number of cases, thereby also playing a guiding role. The discovery of a subclass of miRNA (and miRNA*), whose duplex structure in the pre-miRNA tends to be perfect, preferentially sort into Ago2 brings additional complexity and reveals a new layer of miRNA functions. miRNA often display highly specific expression patterns that reflect important regulatory functions and many reports stressed their crucial role in animal development and homeostasis. These aspects are extensively documented in Drosophila as well. Considerable attention is now given to the ideas that miRNAs are involved in the maintenance/differentiation balance in stem cells and that perturbation of miRNA expression is linked to oncogenesis5.
- The 21 nt siRNAs originate from the processing by Dcr-2 and its partner R2D2 of long dsRNA precursors, such as those produced by inverted-repeat (IR) transgenes. In contrast to most miRNA duplexes, siRNA duplexes are perfect. This structural feature directs loading of single stranded siRNAs into Ago-2, the “RNAi” Argonaute. siRNAs guide cleavage by Ago-2 of target mRNAs with perfectly complementary sequence matches, another feature that differentiate siRNAs from miRNAs.The first physiological function assigned to RNAi was antiviral defense. Well before insect studies, the ability of plants to resist viral infection was associated to the production of small interfering RNAs by the infected host. Viral siRNAs are produced by plant Dicer-like enzymes from double-stranded replicative forms of RNA viruses, or from secondary structures of viral mRNAs. They are loaded into plant Argonautes and direct cleavage of complementary sequences, leading to Viral Induced Gene Silencing (VIGS). With the demonstration that infection of Drosophila S2 cells by Flock House Viruses (FHV) induces a Dcr-2 dependent production of 21nt siRNAs, the antiviral RNAi paradigm began to spread into the insect field. Over the past years, the siRNA pathway (Dcr-2/Ago-2) was shown to be a major axis in the fly’s defense against insect viruses. In addition, endogenous siRNAs (endo-siRNAs) were recently described in flies. They are produced in a Dcr-2 dependent manner from 3 types of loci: cis-natural antisense transcripts, genes with long inverse-repeat structures and bidirectionally transcribed transposon elements. They all produce dsRNA substrates for Dcr-2 and give rise to cis-NAT-, structured- and TE-endo-siRNAs, respectively. There is no clear evidence yet for a physiological function of cis-NAT- and structured-endo-siRNAs. In contrast, it was established that, besides piRNAs, TE-endo-siRNAs contribute to the post-transcriptional silencing of TEs from which they derive. It is noteworthy that they perform this function without the help of piRNAs in somatic tissues, which are virtually devoid of piRNA machinery.
Deep insights into how siRNAs can direct histone modifications and heterochromatin silencing came from studies in S. pombe. In this unicellular eukaryote, sense and antisense transcription of pericentromeric loci gives rises to Dicer-1-dependent production of siRNAs, which direct the Argonaute Ago-1 to cleave transcripts associated with these loci. Targeted transcripts provide templates for an RNA-dependent RNA polymerase (RdRP) to produce more dsRNA, which in turn provide additional substrate for siRNA production in a feed-forward amplification loop. siRNA associated to the RNA Induced Transcriptional Silencing (RITS) complex and the SHREC complex direct histone H3 methylation of lysine 9 by Clr4, a Su(var)3.9 homolog. This allows recruitment of the heterochromatin protein SWI6 (a HP1 homolog), which eventually nucleates and/or maintains heterochromatin 15.
As in S. pombe, Drosophila heterochromatin is prominent in pericentromeric regions, is mostly comprised of TE repeats, and is associated with histone H3K9 methylation by Su(var)3-9, which recruits the heterochromatin protein HP115. Despite these analogies, the evidence supporting a role for small RNAs in heterochromatin formation and transcriptional gene silencing in Drosophila remains indirect: mutants for the Argonautes Piwi and Aubergine or for the RNA helicase Spindle-E exhibit decreased H3-K9 methylation, altered recruitment of HP1 and decreased silencing of heterochromatin markers and of several classes of TEs14,16-19. In addition, it is noteworthy that these data point to piRNAs that are mostly produced in gonads, suggesting that this class of small RNAs play an initiator role in heterochromatin establishment in the germ line.
The new class of endo-siRNAs produced from endogenous double-stranded RNA (dsRNA) precursors was shown to mediate TE silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation, as has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, we used the viral suppressors of RNA silencing B2 and P19. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. We hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3–9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. Our findings provided evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila.
We are further exploring the function of the siRNA pathway in chromatin dynamics. To this aim, we seek for siRNA activity in the nucleus, analyze how they populate loci subjected to heterochromatin silencing and determine their functional relationships with heterochromatin components such as HP1 and Su(var)3.9. In parallel, we analyze the role of siRNA pathway components in the nucleus, including Dicer-2, R2D2 and Argonaute-2. We are also developing computing based approaches for mining of high throughput sequencing datasets in order to analyze at genome-wide level how complex small RNA regulatory networks impact genetic and epigenetic programs.
To study the miRNA pathway, we engineered a reporter system based on the silencing of the GFP by artificial miRNAs. In a genome-wide RNAi screen, this so-called automiR reporter allowed the identification of genes involved in miRNA silencing pathway. Using the automiR reporter, we also identified small chemical compounds that strongly inhibit miRNA silencing activity.
Using LNA-based microarrays and small RNA deep sequencing, we profiled Drosophila miRNAs during metamorphosis. This approach identified miR-282 as a miRNA induced by the steroid hormone ecdysone at the onset of the pupal stage. Moreover, miR-282 loss-of-function mutations arrest development during metamorphosis. Hence, our results provide evidence that in some instances a single miRNA has crucial functions in development.