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Teaser, summary, work performed and final results

Periodic Reporting for period 2 - RegulRNA (Modulation of RNA-based regulatory processes by viruses)


Our understanding of the importance of non-coding (nc) RNAs in various biological processes has grown exponentially in the past few years. Because they are so preponderant, it is no wonder that virtually every biological function is at one point or another regulated by these...


Our understanding of the importance of non-coding (nc) RNAs in various biological processes has grown exponentially in the past few years. Because they are so preponderant, it is no wonder that virtually every biological function is at one point or another regulated by these molecules. Although they vary a lot in their origin, biogenesis and mode of action, we can broadly distinguish between two types of ncRNAs based on their size. At one end of the spectrum are small ncRNAs, which are mostly in the range of 20-30 nucleotides (nt), on the other side, we can find long ncRNAs, which are every other RNAs with a size arbitrarily defined above 200 nt. Ever since research on ncRNAs has started, their importance in the context of viral infection has been the subject of intense scrutiny. Indeed, the very nature of viruses makes them strictly dependent on a host cell to be able to replicate, translate their mRNAs, and ultimately generate new viral particles to spread to the next host. In the frame of this project, we study how a viral infection can impact pathways involving small ncRNAs. The viruses we study are associated with a number of diseases and constitute a threat to human health. There are two types of viruses that are the focus of our research: i) DNA viruses, such as the oncogenic Kaposi’s sarcoma herpesvirus (KSHV) or the mouse cytomegalovirus (MCMV), and ii) arthropod-borne RNA virus, such as Sindbis virus or Chikungunya virus. The results of our research will help us to better understand the molecular mechanisms at play during the viral replication cycle and how the infected organism responds to the infection at the cellular level. Eventually, we might be able to design new antiviral therapeutic approaches to fight some of these pathogens.
The objectives of our proposal are multifold and pertain either to the understanding of the regulation of one family of small RNAs, coined microRNAs (miRNAs), during viral infection, or to decipher the importance of a mechanism of antiviral defense relying on the generation of small interfering RNAs (siRNAs) by the cell. The three main work packages of the project are:
1. To understand how the biogenesis of virus-encoded miRNAs can be regulated by specific cofactors that can bind to the RNA molecule to modulate its processing by ribonucleases Drosha and Dicer
2. To decipher the molecular machinery at play in the degradation of mature miRNAs, and to identify their roles in viral infections
3. To resolve the long-standing question of the antiviral role of RNA silencing in mammals by identifying regulators of this activity

Work performed

We have made good progress in WP1 and 3, and we are now close to getting publishable results for these. We have experienced some delays in WP2 but have recently obtained promising and unexpected results that we are working hard to confirm. Here are the details of the main results obtained per work package.
-WP1. Regulation of miRNA biogenesis
The biogenesis of miRNAs requires the sequential action of two type III ribonucleases, Drosha and Dicer, on a long primary transcript (the pri-miRNA) to give rise to an RNA duplex. One of the two strands of this duplex is then assembled into a protein from the Argonaute family to form the functional mature miRNA that can act on target RNAs. This process is highly regulated at every step, and some proteins have been shown to act by binding directly to the pri-miRNA, or by interacting with Drosha or Dicer to modulate their efficiency. In this project, we have set to study the regulation of miRNA biogenesis using as a model a pri-miRNA transcript of viral origin. Namely, we are working with KSHV, which genome contains a unique cluster of twelve miRNAs grouped on a single pri-miRNA. Although all KSHV miRNAs derive from the same transcript, the relative abundance of each individual mature viral miRNA varies a lot, which indicates that some post-transcriptional regulation takes place. The first part of this WP was dedicated to the analysis of the importance of the secondary structure of KSHV primary miRNA transcript in its cleavage efficiency by Drosha. As of now, we have not yet started this analysis, but we are generating a cell line knocked-out of Drosha, which is an essential pre-requisite to go on with this part.
We have however made very good progress regarding the second part of this work package, which consists in a proteomics analysis of putative proteins binding individual KSHV miRNA stem loop precursors (pre-miRNA). We have designed a novel approach to pull-down proteins (see Research and technological achievements) binding to synthetic pre-miRNA of either viral or cellular origin. We now have data from more than eighty mass spectrometry runs that we are in the process of validating. We found at least one specific proteins for each KSHV miRNAs that should allow us to explain how the biogenesis of viral miRNAs is regulated. As soon as we have validated the role of these proteins, we will submit a manuscript for publication.

-WP2. Interplay between miRNA and long ncRNA and mechanism of miRNA decay.
One pre-requisite for this WP was to identify novel examples of cellular miRNA regulation upon virus infection. We decided to look at this using a slightly different approach, i.e. identifying first cellular miRNAs that can regulate virus infection. Indeed, we previously found one cellular miRNA, miR-27, that could down-regulate the mouse cytomegalovirus (MCMV), and later showed that the virus counteracts by inducing degradation of this miRNA. In order to get a broader picture of the involvement of cellular miRNAs during viral infection, we have performed a genome wide screen to look at the effect of over-expressing or inhibiting each human miRNA individually on viral infection. We made use of an engineered Sindbis alphavirus (SINV) expressing GFP to easily monitor the impact on virus level. Although we did not find new evidence of cellular miRNAs negatively regulating SINV, we found a handful of miRNAs that positively regulate this virus. Very interestingly, among these was the neuron-specific miR-124. Given that SINV can sometimes be linked to encephalitis, this is a very promising finding. We are currently working toward the elucidation of the molecular mechanism through which miR-124 can positively regulate SINV, and we also got preliminary evidence that it can also act on Chikungunya virus. We plan to submit these results for publication before the end of the year.
In addition to this, we have continued our work on the functional characterization of the mechanism behind target RNA depe

Final results

As detailed above, we have made significant progress in all the different parts of this project. The novel findings that go beyond the state of the art are:
-The comprehensive identification of proteins binding specifically to virus-encoded miRNA precursors to regulate their processing. This is especially relevant since one of KSHV miRNA is a well-known orthologue of the oncogenic cellular miRNA miR-155, and it is of prime interest to understand the specificity of expression of these two related miRNAs. In addition, it should also help us to understand whether viral miRNAs really are completely undistinguishable from cellular ones when it comes to their maturation by the cell machinery. By the end of the project, we will have published a complete list of proteins regulating KSHV miRNA biogenesis and will have fully characterized the mode of action of some of them. We will also be able to understand the importance of pri-miRNA secondary and tertiary structure for its recognition by these RNA binding proteins.
-The identification of the neuron-specific miR-124 as a proviral miRNA during alphavirus infection. Until now, there was only very limited evidence that cellular miRNAs could play positive roles for viruses, and our findings indicate that the tissue specificity of some miRNAs could also dictate the tropism of specific viruses. By the end of the project, we will have nailed the molecular mechanism underlying this effect and initiated the exploitation of this finding to design antiviral therapeutics.
-Finally, the determination of interactors of the Dicer protein during viral infection indicates that there are still new functions of this protein to be discovered. We are very near to complete the characterization of these interactions and to decipher their functional implication during infection. We have also started to extend these observations to other viruses, such as Dengue or Zika virus. The unbiased approaches that we have launched are also bringing new answers regarding the sensing of dsRNA generated during viral infection, and until the end of the project we will dramatically have improved our understanding of the mechanisms at play in the initial steps of a virus infection.