RNA INTERFERENCE PATHWAY

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Stress and transposition

Living organisms permanently encounter stress such as variation in climatic factors, interaction with other organisms, presence of toxins or chemicals. A distinction between biotic and abiotic stress can be done. Biotic stress is the one caused by another organism, while abiotic stress is produced by environmental factors such as sunlight, humidity and wind. In order to survive organisms need to adapt to stress through tolerance or resistance (107, 108).
The re-organization of the genome induced by TEs movements can play an essential role in host response to stress, facilitating the adaptation of populations and species facing changing environment. For example, Gonzalez et al. 2008 (109) showed that fly adaptation to climate was related to TEs insertions. As mentioned above, the allele of the gene CHKov1 where the Doc1420 element is inserted induces resistance to Sigma virus infection (101). And one insertion in the gene Cyp6g1 provides insecticide resistance to the flies (110). All these examples put in evidence the importance of TEs insertions in the acquisition of new gene functions for insect survival.

Transposon defense: a way to survive

Host-transposon arms race

The balance between TEs genome defenses and TEs damage confer an important role of TEs in evolution and gene regulation of the host organism (111, 112). In this arms race different mechanism to regulate TE expression exist.
To move and invade the host genome TEs need the production of proteins. TEs can be autonomous, producing their own proteins (113, 114), or non-autonomous, when they require the production of proteins from the cell or from other TEs for their movements (115). These proteins production is the limiting step for the replication and the spread of transposons. One interesting example comes from the P-element in Drosophila: P-element contains three introns. Two of them are spliced ubiquitously, whereas the third intron is only spliced in the germline and is necessary for the production of the full length transposase, restricting the P-element activity to germline only (116).
On the other hand, TEs are regulated by the host defense mechanisms against transposition. Whereas natural selection is widely considered as the dominant force limiting TE proliferation (117), the arms race between TEs and the host genomes has driven the evolution of the recently discovered Piwi-interacting RNA (piRNA) and endogenous small interfering RNA (endo-siRNA) pathways, which have profound impacts on gene regulation and epigenetic silencing of TEs (see below) (118-121).

Several defenses, one mechanism: RNA interference

As mentioned earlier, insects face threats during their life and, in order to survive, they need to defend themselves against these menaces. RNA interference (RNAi), a branch of the innate immune response in insects, is a biological mechanism guided by small RNA molecules (from 21 to 30 nt) enabling the sequence-specific recognition of cognate nucleic-acid target sequences and their degradation, translational arrest or transcriptional regulation. In insects, RNAi-based responses mediate robust antiviral defense and protection against transposition.
In the next section I will describe the different RNAi pathways found in insects and their involvement in defense.

RNA interference pathway

miRNA

MicroRNAs were the first class of small RNAs to be discovered. They were identified by Lee et al. in 1993 (122) in the nematode C. elegans. Today we know that miRNAs are found in all kingdoms, and that they are highly conserved among them (123-125). The main function of miRNAs is to regulate host gene expression by initiating the degradation of their targets or by blocking their translation (125, 126). Their activity is essential in the regulation of organ development, cellular differentiation and homeostasis (127, 128). Mutations in this pathway disrupt development and often lead to embryonic lethality (129, 130).
The miRNA pathway (Fig. 5B) is initiated by the expression of genome-encoded miRNA gene transcripts. These primary miRNAs are capable of folding back on themselves to form one or more dsRNA stem-loop structures that trigger the pathway. The primary miRNAs are processed in the cell nucleus by a protein complex formed by Drosha and Pasha to produce the precursor miRNA, which is exported to the cytoplasm (131, 132). Precursor-miRNAs are then further processed into 21- to 23-nt small dsRNA (miRNA) duplexes by another enzymatic complex formed by Dicer-1 (Dcr-1) and Loquacious (LOQS)-PA or LOQS-P  (133, 134). The miRNA duplex produced in this reaction is loaded into the AGO1-containing RNA-induced silencing complex (RISC). One strand of the duplex, the miRNA*, is released from the complex and quickly degraded, forming a mature RISC that contains only one small RNA strand (135). RISCs harboring miRNAs primarily target protein-coding mRNAs, producing either translational inhibition or mRNA degradation. Target recognition by miRNA does not require perfect homology. The miRNA pathway is active in both somatic and germline tissues.

siRNA

Small interfering RNA (siRNA) are small RNAs with a variable length depending on the organism (21-24 nt) that can bind specifically to RNA and restrict gene expression via mRNA cleavage. The siRNA pathway was discovered in 1990’s in plants, when Napoli and Jorgensen (82) overexpressed the enzyme chalcone synthetase (CHS) and obtained white flowers instead of the purple ones expected. This discovery lead, during the years that followed, to unravel the molecular basis and functions of the siRNA pathway (136, 137).
The siRNA pathway (Fig. 5A) can be triggered in cells by either endogenous or exogenous dsRNA molecules. Endogenous dsRNA molecules are produced from long genomic transcripts capable of forming extensive fold-back structures or double-stranded regions generated by intermolecular hybridization of overlapped transcripts (138, 139). Exogenous dsRNA molecules can be derived from any environmental source, such as viral dsRNA molecules. In the siRNA pathway, dsRNA is recognized and processed in the cytoplasm by Dicer-2 (Dcr-2) into 21-nt siRNA duplexes harboring 2-nt 3′ overhangs (140). After being diced, siRNA duplexes are loaded into the Ago-2-containing RISC. The biogenesis and loading of siRNA duplexes into the RISC require the activity of LOQS and R2D2 as Dcr-2 cofactors. The LOQS-PD isoform and R2D2 are required for the production of siRNAs derived from endogenous dsRNA triggers, and R2D2 is primarily recruited in the production of virus-derived siRNAs (vsiRNAs) (141, 142). Once loaded into the RISC, one strand of the siRNA duplex, termed the passenger strand, is eliminated from the RISC. The single-stranded siRNA that remains in the RISC, termed the guide strand, is then 2′-O-methylated at its 3′-terminal nucleotide by the RNA methyltransferase DmHEN1 (143, 144), resulting in a mature, active RISC. Sequence-specific recognition mediated by the retained siRNA guide strand, which requires complete complementarity, then induces target RNA cleavage via the slicing activity of Ago-2. Although endogenous siRNA targets are mostly transposons and protein-coding mRNAs, vsiRNAs recognize virus-derived sequences. As with the miRNA pathway, the siRNA pathway is ubiquitously expressed.

piRNA

A third RNA interference pathway, the piwi-interacting RNA pathway (piRNA) was recently described (Fig. 5C). Molecules initiating the piRNA pathway are ssRNA precursors transcribed from chromosomal loci that mostly consist of remnants of transposable element sequences, called piRNA clusters (111). Biogenesis of piRNAs involves two steps, primary processing and secondary amplification. Production of piRNAs is Dicer independent and mainly relies on the activity of PIWI proteins, a subclass of the AGO family (145). Primary piRNAs are processed from ssRNA transcripts derived from piRNA clusters. Zucchini endonuclease (Zuc) cleaves primary piRNA precursors and generates the 5′ end of mature piRNAs (146-148). The cleaved precursor is loaded into PIWI or Aubergine (Aub) proteins and then trimmed by an unknown nuclease to reach its final length. After trimming, piRNAs undergo a final 3′-end 2′-O-methyl nucleotide modification catalyzed by DmHEN1 (143, 144) to yield mature piRNAs. Primary piRNAs harbor a 5′ uridine bias (U1) (149). Cleavage of the complementary active transposon RNA by primary piRNAs loaded into Aub proteins initiates the second round of biogenesis, which leads to the production of secondary piRNAs that are loaded into Ago-3. During this ping-pong, or amplification, cycle, Aub and Ago-3 proteins loaded with secondary piRNAs mediate the cleavage of complementary RNA, generating new secondary piRNAs that are similar in sequence to the piRNA that initiated the cycle. The complementary secondary piRNAs usually have a 10-nt overlap and contain an adenine at position 10 (A10) (150). Most data indicate that the piRNA pathway is mainly active in germline tissues, where it acts as a genome guardian by cleaving transposons RNA or transcriptionally silencing transposable elements.
(A) Exo and endo-siRNA pathway: dsRNAs are processed by Dicer-2 (Dcr-2) and its co-factor R2D2 for exo-siRNA or LOQ-S for endo-siRNA, and generates siRNA duplexes. This complex loads the siRNA duplex into Argonaute-2 (Ago-2) protein. The passenger strand is unwound and released, the guide strand stays into Ago-2 and its 3’ extremities are protected by the addition at the 3’ end of a 2’-O-methyl modification catalyzed by the methyltransferase HEN1.
(B) miRNA pathway: miRNA genes are transcribed into a primary miRNA (pri-miRNA) transcript, which is cleaved by Drosha and Pasha complex to give a short miRNA precursor (pre-miRNA). This pre-miRNA is exported to the cytoplasm, where it is processed by Dicer-1 (Dcr-1) and LOQ-S to generate a miRNA duplex. The duplex is loaded on Ago1-RISC complex. One strand, the miRNA*, is released. The other strand, the miRNA, guides translational repression of target RNAs.
(C) piRNA pathway: piRNAs of 24-29 nucleotides long are derived from a ssRNA precursor, a piRNA cluster. In the primary processing, piRNAs coming from the cleavage of the piRNA precursor are processed and loaded on PIWI protein in the cytoplasm. Then the 5’ extremities of piRNAs will be matured by the Zucchini (Zuc) protein and its co-factors. The methyltransferase HEN1 adds at the 3’ end the 2’-O-methyl modification. The secondary processing, the amplification cycle, generates additional piRNAs. Antisense piRNAs are loaded in PIWI or Aub, while sense piRNAs are loaded in Ago-3.
S-adenosyl methionine (SAM); S-adenosyl homocysteine (SAH) ; Polymerase II (pol II). RNA interference: protection against the non-self miRNAs, gene expression and immunity During a viral infection, cells, tissues and entire organisms need to develop a defense strategy. This results in modifications in the expression of genes involved in immunity and in different cellular processes. Since miRNAs are gene regulators, their potential role in immunity was long suspected and, indeed, miRNAs protect the infected host against the non-self through the modulation of the self. miRNAs can be produced from both host and virus. We observe host miRNAs, which regulate viral transcript (151), viral miRNAs that regulate host transcripts (152, 153) and viral miRNAs that can regulate viral transcripts via the host miRNA machinery (154).
miRNA regulation can impact different cellular processes and thus change many factors that influence viral infection. For example in mosquitoes, studies showed that miRNA expression influence the viral tropism of certain viruses. For example, in Ae. aegypti miR-275 depletion affects egg production and blood digestion, two important mechanisms for virus life cycle and transmission (155). miRNA can also directly impact immune pathways. For example, Ae. aegypti miR-375, detected after blood meal, targets the 5’UTRs of the Toll immune pathway components Cactus and REL1 (156). Other examples of miRNA gene regulation during host-pathogen interactions were observed in cases of arboviruses infections in mosquitoes. The expression in vitro of the KUN-miR-1 during West Nile infection, up-regulates the GATA4 mRNA and induces protein accumulation that supports viral replication (152).
During DNA virus infection miRNA produced by the virus can induce viral cycle modifications, as for Nudivirus-1 pag1 miRNA, which down regulates the viral early gene hhi1 to induce viral latency (157).
The major issue in the battleground is the detection and neutralization of outsiders. In all organisms the crucial step for viral defense concerns the recognition of self versus non-self. Viral dsRNA is key for the detection of a viral infection. As a replicative intermediate of all known viruses (except retroviruses), its recognition by the exo-siRNA pathway contributes to the defense system of insects (28, 29, 158). This recognition step will lead to the degradation of the viral genome in the cytoplasm of the infected cells, and allows in some cases the clearance of the virus. Several evidences suggest the importance of RNAi in diptera antiviral immunity. First, flies with mutations in known RNAi pathway components are hypersensitive to RNA virus infections and develop a dramatic increase in viral load (28, 29, 158); second, many insect viruses, encode suppressors of RNAi that counteract the immune defense of the insect (159-161). Finally, the rapid evolution of RNAi pathway genes compared with miRNA pathway genes in Drosophila also suggests an ongoing arms race between insect viruses and hosts, and highlight the importance of RNAi as antiviral defenses (162).
piRNAs and endo-siRNAs, insiders recognition and control As previously described, TEs represent threats for insect viability and species sustainability. Therefore a good protection system is important to avoid genome invasion. piRNAs are the genome guardians. Indeed, the piRNA pathway is restricted to the germline and regulates the accumulation of TEs to avoid their transmission to the progeny (111). Furthermore the piRNAs synthetized in the germline are maternally transmitted and allow the protection of the genome during the development stage (120, 163), constituting an inherited defense.
During many years it was assumed that TE movements were restricted to germline, but recently transposition in somatic cells was also observed (121, 164). Insects have selected different silencing mechanisms for somatic and germline TEs. In a general manner, endo-siRNAs have been privileged to silence TEs in somatic cells while piRNAs do it in germline cells (138).

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Innate immunity strategy: the flexibility

The observation that RNAi pathways are conserved across eukaryotes means that the common ancestor of these organisms had a functional RNAi pathway billion years ago (165, 166). Its conservation highlights the importance of the RNAi process on the adaptation to different threats. This ability relays, in part, on the evolutionary rate of the Argonaute proteins, the core component of the RNAi machinery. Indeed, Lewis et al. 2016 (167) showed not only that Drosophila Ago-2 and Ago-3 proteins have a high evolutionary rate, but also that the gene turnover (number of gains and losses per million years) of Ago-2 and Piwi/Aub are important. These observations along with other studies where duplications of Argonaute and Piwi proteins are observed in the diptera clade (167-170), confirmed a high selective pressure on these proteins. In a more global approach Argonaute proteins with diverse functions and different copy number were found across different eukaryotic clades (171), illustrating the dynamism of their evolution. All these duplication events possibly drive the acquisition of new functions for RNAi proteins, as exemplified for the involvement of the piRNA pathway in antiviral response in mosquito (172-174) besides the classical transposition control role.

Outline of this thesis

In insects, especially in Diptera, it was observed that different small RNAs pathways play a role in the defense against different genome parasites such as viruses and TEs. This work focuses on the involvement of the piRNA pathway on antiviral defense in Drosophila melanogaster. In chapter 2, I describe the protocol to homogenize the genetic background of the different mutant flies used in my studies. The chapter 3, presents the main results of my research concerning the study of viral piRNAs in diptera. I observed that, unlike mosquitoes, Drosophila melanogaster does not produce viral piRNAs, independently from the type of viruses, the infection state or the viral transmission route. I demonstrate that adult Drosophila melanogaster flies do not require the production of viral piRNAs to mount an efficient antiviral response. Following these results, I investigate whether the production of piRNAs are affected by stress due to viral infection, and this is the subject of Chapter 4.
Finally, chapter 5 is dedicated to a general discussion on the results of this thesis and some perspectives to understand TEs impact during infection in Drosophila melanogaster.

Table of contents :

GENERAL INTRODUCTION
THE INSECT’S INVADERS
THREATS FROM OUTSIDE
THREATS FROM INSIDE
SEVERAL DEFENSES, ONE MECHANISM: RNA INTERFERENCE
RNA INTERFERENCE PATHWAY
RNA INTERFERENCE: PROTECTION AGAINST THE NON-SELF.
INNATE IMMUNITY STRATEGY: THE FLEXIBILITY
OUTLINE OF THIS THESIS
BIBLIOGRAPHY
ON THE IMPORTANCE OF FLY GENETIC BACKGROUND
PROTOCOLS OVERVIEW
VIRUSES CHECKING AND CLEANING
CONTROL OF WOLBACHIA INFECTION IN FLIES
THE GENETIC BACKGROUND: INTROGRESSION PROTOCOL
THE PIRNA PATHWAY MUTANT FLIES: SELECTION, CHARACTERISTICS AND ROUTINE WORKS.
BIBLIOGRAPHY
THE PIRNA PATHWAY IS NOT REQUIRED FOR ANTIVIRAL DEFENSE IN DROSOPHILA
MELANOGASTER
VIRAL INFECTION, TRANSPOSON EXPRESSION AND PIRNAS
DISCUSSION & PERSPECTIVES
A DIRECT ROLE OF THE PIRNA PATHWAY IN ANTIVIRAL DEFENSE IN DIPTERA ?
INDIRECT ROLE OF THE PIRNA PATHWAY IN ANTIVIRAL DEFENSE IN DROSOPHILA
BIBLIOGRAPHY

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