Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - D-FENS (Dicer-Dependent Defense in Mammals)

Teaser

Viral infection or expansion of mobile genetic elements (retrotransposons) in the genome often result in production of double-stranded RNA (dsRNA). dsRNA can be intercepted by RNase III Dicer acting in the RNA interference (RNAi) pathway, an ancient eukaryotic defense...

Summary

Viral infection or expansion of mobile genetic elements (retrotransposons) in the genome often result in production of double-stranded RNA (dsRNA). dsRNA can be intercepted by RNase III Dicer acting in the RNA interference (RNAi) pathway, an ancient eukaryotic defense mechanism, which remains a key innate immunity pathway in invertebrates and plants. In mammals, RNAi has been functionally replaced by the interferon pathway, except of mouse oocytes, where it is an essential pathway regulating gene expression. However, the endogenous mammalian RNAi appears rather asleep than lost and its physiological role(s) in mammalian cells remain poorly understood. A factor underlying mammalian RNAi dormancy is inefficient processing of dsRNA by Dicer, an enzyme, which initiates RNAi by processing long dsRNA into small RNA molecules. Yet, a simple shortening of Dicer by removing its N-terminal helicase domain makes a highly active enzyme and hyperactive RNAi, which is naturally found only in mouse oocytes because of a unique mutation, which occurred during mouse evolution.

The project D-FENS focuses on a general and important biological process: How mammalian cells can deal with threats associated with dsRNA in order to maintain viability and genome integrity? Understanding principles governing evolution of RNAi and co-existence of RNAi with evolutionarily recent antiviral pathways could bring new strategies for antiviral therapies. Understanding evolution of genome maintenance and impact of mobile elements on rodent genomes means understanding fundamental principles of evolution of new gene regulations and functions.

A somewhat unexpected but outstanding model for studying dsRNA effects and RNAi is the mammalian oocyte despite the scarce material poses limits for some experimental approaches. First, an oocyte must efficiently suppress retrotransposons to prevent transmission of new insertions into future generations – the pressure on effective retrotransposon suppression is thus higher in oocytes than in somatic cells. Second, oocytes frequently evolve unique adaptations not found in cells of the body (such as the truncated Dicer variant in mouse oocytes), which may bring rare insights into to a molecular mechanism. Third, RNAi and related mechanisms function in mouse oocytes in an apparent absence of the interferon response. Fourth, remarkable progress in gene expression analysis and genetic manipulations make oocytes a more accessible model system, allowing for performing experiments nowadays, which were extremely labor- and time-consuming a few years ago.

The D-FENS project will use genetic mouse and hamster models to define common, cell-specific and species-specific roles of RNAi in mammals. Understanding evolution of mammalian RNAi requires systematic analysis of miRNA and piRNA pathways, two related molecular mechanisms employing small RNAs in gene regulation and retrotransposon suppression in the germline, respectively. As the three pathways are closely linked, one has to study evolution of RNAi in the context of the other two pathways. Accordingly, the project has three major objectives:

(I) Explore consequences of hyperactive RNAi in vivo. While mouse viruses co-evolved with mice to adapt to and overcome their immune system, they were not adapting to RNAi because they to the interferon response instead of RNAi. We will produce a genetically modified mouse expressing a truncated highly active Dicer enzyme in all cells. This will reveal level any negative effect of hyperactive RNAi on the whole organism, the relationship between RNAi and mammalian immune system, and potential of RNAi to suppress viral infections in mammals.

(II) - Define common and species-specific features of RNAi in the oocyte. Functional and bioinformatics analyses in mouse, bovine, and hamster oocytes will define rules and exceptions concerning endogenous RNAi roles, including RNAi contribution to maternal mRNA degradation and co-existence with the miR

Work performed

(I) Short Dicer variant & antiviral potential of RNAi

Objective of this part is to explore consequences of hyperactive RNAi in vivo. While RNAi is the main antiviral pathway in invertebrates, such a role in mammals is unclear while the canonical pathway activity in somatic cells is negligible if not absent. We proposed to approach the issue of RNAi potential by producing a “super RNAi” mouse model where the endogenous Dicer gene would be modified to express an equivalent of a shortened Dicer isoform naturally found in mouse oocytes. During the first half of the project, we identified in cultured cells for a functional highly active RNAi. The cell lines created during this work are also studied with respect to antiviral immunity in vitro, however, so far we did not observe an inhibitory effect on coxackie viruses, our model RNA viruses, so far. The essential milestone of has been production of mice expressing the shorter Dicer enzyme. Such animals would make an excellent model to test hypotheses concerning evolution of RNAi and its antiviral role in mammals. During the first half of the project, we have been struggling with producing the desired genetic modification in mice. We finally succeeded by the end of 2017. At the moment, we are breeding the mice for further studies. Multiple breedings are needed to clean up the genetic background from possible mutations introduced along with the desired modification. Importantly, we also managed to produce a control mouse model, which is completely devoid of the shorter Dicer variant while its genome was modified in a similar way as the shorter variant-expressing mouse mentioned above.

In terms of bioinformatics analysis of Dicer, we made a major progress. We produced and started to analyze transcriptomes of bovine and rodent oocytes, which provide an excellent collection for studying evolution of gene regulation in the mammalian female germline. Remarkably, during bioinformatic work on annotation of murine long non-coding RNAs (lncRNAs) in another project, we stumbled upon (1) lncRNAs, which are substrates for siRNAs, which regulate gene expression and (2) massive contribution of MT retrotransposon family LTR insertions to mouse transcriptome remodeling. This work lead to an article covering impacts of MT retrotransposons on maternal transcriptomes and evolution, which was published in the Genome Research journal in 2017:

Franke V, Ganesh S, Karlic R, Malik R, Pasulka J, Horvat F, Kuzman M, Fulka H, Cernohorska M, Urbanova J, Svobodova E, Ma J, Suzuki Y, Aoki F, Schultz RM, Vlahovicek K, Svoboda P. Long terminal repeats power evolution of genes and gene expression programs in mammalian oocytes and zygotes. Genome Res. 2017 Aug;27(8):1384-1394. doi: 10.1101/gr.216150.116.

This article shows that endogenous RNAi in mouse oocytes did not form just thank to a single etrotransposon insertion into the Dicer gene but that many distinct insertions of one family (MT) retrotransposons shaped repeatedly the entire endogenous RNAi pathway at the level of substrates, small RNA biogenesis and silencing effects. Dicer gene in the mouse lineage has been actually functionally remodeled twice.

In addition, we also wrote two review articles, one summarizing mammalian Dicer biology and one impact of retrotransposons on evolution and function of lncRNAs:

Svobodova E, Kubikova J, Svoboda P. Production of small RNAs by mammalian Dicer. Pflugers Arch. 2016 Jun;468(6):1089-102

Ganesh S, Svoboda P. Retrotransposon-associated long non-coding RNAs in mice and men. Pflugers Arch. 2016 Jun;468(6):1049-60

Because of delayed “RNAi mouse” production, analysis of antiviral effects in genetically modified mice at the Slovak Medical University (SMU) did not take place yet. The plan is that SMU will perform the analysis of RNA-mediated antiviral response using a coxackie virus model. The work there is supervised by assoc. Prof. Shubhada Bopegamage, MSc. and is coordinated via Skype meetings. Because of th

Final results

The project has several state-of-the-art aspects. Since the most investigated mammalian RNAi model is the outstanding mouse case, we know little about general and species-specific features of endogenous RNAi in mammals. The proposed experiments in mouse, hamster and bovine oocytes represent leading research of endogenous RNAi in mammals. This research will be supported by newest genetic engineering tools represented by guided nucleases (TALENs and more recent CRISPRs).

For example, we will use these nucleases will restructure Dicer gene exon/intron structure while tagging the expressed protein. This will produce an animal model to test in vivo whether hyperactive RNAi 1) improves antiviral response, 2) can substitute for the piRNA pathway, 3) has any negative effects on the organism. At the same time, this model will address the importance of the N-terminal DExD domain. All this new knowledge will allow to determine whether enhancement of RNAi could be a viable strategy for a new type of antiviral therapy.

Another state of the art is production of a golden hamster knock-out model lacking the piRNA pathway. While at the time of writing this project proposal it would appear as extremely challenging task with an unpredictable outcome, it is likely that at the end of the project, genetic modifications of golden hamster will become a viable option for identifying recently evolved mouse-specific features, which are not shared with the hamster.