C’est lors de ces séances que j’ai pu découvrir le phénomène de silençage de l’ARN et par la même occasion l’équipe de recherche qui excellait dans ce domaine. Un merci spécial à Peter, Lionel et Derrick pour toutes ces réflexions que j'ai pu avoir avec chacun d'eux sur mon projet et leur lumière plus qu'éclairée.
Foreword
They were further characterized by Elbashir and Tuschl and named short interfering (si)RNAs (Elbashir et al., 2001a). The first virus-encoded miRNAs were discovered by the Tuschl laboratory in cells latently infected with Epstein-Barr virus (EBV) (Pfeffer et al., 2004).
Regulation by small non-coding RNAs and miRNAs evolution in
RNA silencing: a conserved process of genetic regulation among eukaryotes
However, this is not a definitive explanation, as some budding yeasts such as Saccharomyces castellii and Candida albicans do have an RNA silencing machinery (Drinnenberg et al., 2009). On the other hand, RNA silencing appears to have become essential in many multicellular eukaryotic lineages, which we can infer through the lethality of Dicer mutations due to the involvement of miRNAs in the regulation of development.
Evolutive origin of miRNAs
- Contrasting miRNA pathways in plant and animals
- A brief overview of canonical miRNA biogenesis in plant versus animal
- Non-canonical miRNAs
- Genomic arrangement of miRNA genes
- Target recognition by plant and animals miRNAs
- A miRNA-like regulation in the last common ancestor of plants and animals?
- Evolutionary mechanisms for de novo miRNAs emergence and target acquisition
- Inverted duplication model
- MiRNAs deriving from transposable elements
- snoRNA and tRNA-derived miRNAs
- De novo generation of miRNA hairpins and random selection model
- Transcriptional control model of new miRNAs
This modification prevents 3'-uridylation of mature miRNAs that accelerates their turnover (Figure 4) (Li et al., 2005). In support of this hypothesis, newly evolved miRNAs have been described in the vicinity of highly conserved animal miRNAs (Berezikov et al., 2011).
Animal miRNAs: biogenesis, mode of action and targeting functions
Biogenesis of miRNAs in mammals
- Generation of the pre-miRNA and export into the cytoplasm
- Dicing of the pre-miRNA into miRNA/miRNA* duplex
- Mature strand selection and loading into RISC
In some cases miRNA*, which is usually degraded, can also be incorporated (Figure 4) (Carmell et al., 2002). Central mismatches promote RISC loading, and thus their properties are reminiscent of Drosophila AGO1 (Yoda et al., 2010).
Mode of action miRNAs in animals
- Target recognition
- Characteristics of animal miRNAs target sites
- Influence of the binding site sequence context
- Assembly of the miRISC-mRNA ternary complex
- Mechanism of miRNA-mediated target repression
Consistently, it has been shown that seed pairing can be both necessary and sufficient for miRNA regulation—repression is inhibited by G:U oscillations in the seed match—and that the miRNA's 3'-pairing region with the site the target is rarely determinative (Brennecke et al., 2005; Doench and Sharp, 2004). Cleavage and subsequent release of the target RNA will then be accompanied by the return of Ago to the extended conformation (Filipowicz, 2005).
Roles of functional interactions of miRNAs with their targets
- The micromanager model
- A role for neutral targets: the pseudotargets
- The competing endogenous RNAs models
The only physiological effects of miRNAs are due to suppression of authentic targets (red). According to this model, the neutral targets defined by Bartel and Chen would fall into the class of pseudotargets, and the shift and tuning targets would represent two subclasses of authentic targets.
Methods for identifying and validating miRNA targets
Predictions methods of miRNA targets
- Computational methods
- Genome-wide RNA and protein expression analyses
- Biochemical methods
The PITA (probability of interaction by target access) algorithm was designed following this consideration (Kertesz et al., 2007). Thus, to obtain a list of putative targets, a bioinformatic filter must be applied (Thomson et al., 2011).
Validation of miRNA targets as biologically relevant targets
- Experimental validation of a predicted miRNA target gene
- Importance of biological function assessment of characterised miRNA-mediated
However, before the actual reporter analysis can be performed, the cloning step for the construction of the luciferase reporters can be quite labor intensive (Kuhn et al., 2008; Thomson et al., 2011). Alternatively, mRNA destabilization can be observed by analyzing its polyadenylation state (Pasquinelli, 2012; . Sallés et al., 1999).
Kaposi’s sarcoma-associated herpesvirus
- The herpesvirus family
- KSHV epidemiology and virus transmission
- KSHV associated diseases
- Kaposi’s sarcoma
- Primary effusion lymphoma
- Multicentric Castleman’s disease
- Experimental systems for the study of KSHV infection
- Animal Models
- Cell culture systems
- Genetic analysis
- Viral replication
- Genome structure and organisation
- Overview of the viral cycle
- Model for viral oncogenesis
- General considerations on oncogenic viruses
- Mechanisms of KSHV-induced oncogenesis
Attempts to grow in vitro infected tumor cells directly from KS lesions have been unsuccessful, resulting in the loss of the virus after a few passages. This study therefore provides the first animal model, which significantly recapitulates the important aspects of KSHV infection in humans, and will enable a better understanding of the disease's physiology (Chang et al., 2009). In latency, viral expression is severely restricted and only few of the viral genes are expressed.
Finally, many of the KSHV proteins with important putative tumorigenesis activities are expressed only in a small subset of KSHV-lytically-infected cells.
Virus-host interactions involving cellular miRNAs
This results in the increase of miR-218-target laminin 5"3 (LAMB3) expression and can increase cell migration and tumorigenicity (Martinez et al., 2007). The latter targets p300/CBP-associated factor (PCAF), a proposed cofactor for the HIV-1 Tat transactivator (Triboulet et al., 2007) Another hypothesis that emerged recently is that the binding of miR-122 (and most likely Ago2) at the very 5' end of the genome acts as a protection against cellular factor (Machlin et al., 2011).
Another example of a viral ortholog of the cellular miRNA miR-29a was recently shown for bovine leukemia virus (BLV) (Kincaid et al., 2012).
Virus-encoded miRNAs
- MiRNAs encoded by DNA viruses
- MiRNAs encoded by RNA viruses
- Conservation of viral miRNAs
Consistent with previous studies, the small RNA has not been detected in infected mammalian cells (Hussain et al., 2012). The authors therefore predicted that Pol III-transcribed miRNAs could be a common feature of retroviruses, which will therefore require further studies (Kincaid et al., 2012). This is indeed the case when comparing genomic sequences of different KSHV isolates, for which the miRNAs are generally highly conserved (Marshall et al., 2007).
Therefore, changes in the target site may be the first event followed by a viral miRNA acquiring compensatory mutations to maintain its binding to its target mRNA (Cullen, 209; Walz et al., 2010).
Virus-encoded miRNAs functions
- Models of viral miRNA function
- Non-canonical functions
- Viral targets
- Cellular targets
- In vivo roles
Indeed, no major differences were observed in the response to cytotoxic T cells, in the secretion of the IFN-$ cytokine and in the viral clearance in the experimentally inoculated mice (Sullivan et al., 2009). The same has been observed for the avian infectious laryngotracheitis virus (ILTV) miR-I5, which mediates repression of the ICP4 transactivator ( Waidner et al., 2011 ). However, in this case, direct targeting by the miRNA has not yet been demonstrated (Iizasa et al., 2010).
This regulation has been formally shown to be directly responsible for the induction of HzNV-1 latency (Wu et al., 2011).
Identification
In the following sections, I will only focus on the results I obtained during my thesis published in PLOS Pathogens in an article attached below. One of these studies deals with the identification by RISC-IP of EBV and KSHV miRNAs targets (Dölken et al., 2010a) and is cited and commented on several times in the different parts of this manuscript. The three other articles are outside the scope of the viral miRNAs field, as they deal with the roles of cellular miRNAs in the context of rheumatoid arthritis, and are therefore not further described in this manuscript (Alsaleh et al., 2009; Philippe et al., 2012; Semaan et al., 2011).
KSHV miRNAs coding regions are shown as gray arrows, the two alternative promoters for kaposin transcripts as black arrows, the kaposin transcripts as black lines, and the intronic region in the larger transcript as a break in the line.
Features of the mature miRNA sequences
MiR-15a and -16 are both associated with tumor suppressor activity, and a potential analogous function shared by the virus and the two cellular miRNAs remains to be demonstrated. Moreover, such an analogous function arising from a “shifted” seed identical to the cellular miRNA seed sequence could be possible, as reported by PAR-CLiP for a KSHV-encoded miRNA that partially mimics the cellular function of miR-142-3p. Indeed, miR-K12-10a has variant forms with an additional nt in the 5' (displacing its original seed sequence) that has a 7-mer seed region identical to the 5' one-nt shorter form of miR-142-3p.
Expression patterns
However, variability that could affect processing was observed in some of the pre-miRNAs sequences (Marshall et al., 2007). Pri-miRNA expression is currently reported to always be associated with a transcript that also contains at least the Kaposin CDS (Cai and Cullen, 2006; Pearce et al., 2005). Indeed, Gottwein et al., in their PAR-CLiP study, observed a different distribution of Ago2-loaded viral miRNAs.
Nevertheless, these results still remain good indicators of the differences in the expression levels of KSHV miRNAs, as when considering the star.
Function of KSHV miRNAs and their potential involvement in viral
Function of KSHV miRNAs and their potential involvement in viral oncogenesis The functions of KSHV miRNAs will not be detailed in this section, as many. Cellular targets of viral miRNAs involved in the control of apoptosis and cell survival The targets described in this section are listed in Table 6.
Cellular targets of viral miRNAs involved in the control of apoptosis and
Moreover, genes involved in cell survival have also been described as targets of viral miRNAs, such as the transcriptional repressors BCL6 or BACH1 (Table 6). Targeting such genes involved in cell survival/apoptosis by viral miRNAs makes perfect sense as they may be implicated in antiviral mechanisms via the intrinsic or extrinsic induction of apoptosis, respectively mediated by the cell itself or by extracellular signals (e.g. cytokines , death receptor activation) of cells in the immune system).
KSHV miRNAs target Caspase 3 and regulate apoptosis
The relative abundance of KSHV miRNAs was determined using DG-75-K10/12 small RNA deep sequencing data. For all KSHV miRNAs tested, except miR-K12-9, we could show a strong repression in the presence of pcDNA-K10/12 ( Figure 4A ). The lack of miR-K12-9 activity may be related to its lower expression in the context of the K10/12 construct (Tables S1 and S2).
Dot plot of changes in gene expression observed in DG-75 cells (B) and in Ea.hy926 cells (C).
Critical review of the experimental approach
However, this allowed us to discard part of the observed changes in gene expression that are due to natural variations or, on the other hand, part of the genes that are indirectly repressed by the expression of KSHV miRNAs. The second filter was to look only at those transcripts that contained in their 3'UTR at least one 6-mer seed that matched one of the KSHV miRNAs. Apart from the possibility that protein factors modify some of the miRNA-mediated regulation (and which is not really well known at the moment), most of these hypotheses could be tested using bioinformatics.
Only two of the candidate genes tested showed an interesting significant suppression: CASP3 and RAD51AP1.
Insights from our results
Indeed, during lytic infection, many KSHV proteins are known to play a role in inhibiting apoptosis, so the role of miRNAs in this function may be negligible. Viral miRNAs would then act as a relay of the functions of viral proteins after the termination of their expression caused by the establishment of latency. Furthermore, as detailed in section 5.6 of the introduction, the viral proteins expressed during KSHV latency, for the most part (LANA, LANA-2, vCyc and vFLIP), have been shown to play a direct or indirect role in inhibiting programmed cell death in various levels.
This means that the viral miRNAs target at least one other extracellular factor, which is involved in the caspase-independent induction of apoptosis.
A potential inhibition of CASP3-mediated cleavage of Dicer by viral
A Cellular Function for the RNA Interfering Enzyme Dicer in the Maturation of the Let-7 Small Transient RNA. Critical role of the virus-encoded ortholog MicroRNA-155 in the induction of Marek's disease lymphomas. Therefore, we compared the transcript half-lives of cellular miRNA targets with those of 8,300 genes expressed in BL41 obtained from our recently published atlas of transcript half-lives in human B cells ( Friedel et al., 2009 ).
We next looked at the correlation of the enrichment in the Ago2-IP in BL41. In total, approximately 60% of known KSHV and EBV miRNA targets showed detectable enrichment in our assay (summarized in Table S3B). Therefore, this approach may help decide which of the large number of viral miRNA targets to pursue in further studies.
