Biology of Non-coding RNA

The major focus of my lab is on gene regulation by small RNA molecules acting in RNAi-related pathways. Since their discovery at the start of the 21st century, many different RNAi-related pathways have been identified and it is now evident that although all of these pathways depend on proteins from the Argonaute family, each pathway has its own unique characteristics and effects on gene expression. These can range from relatively minor effects on translation (in the case of many miRNAs) to the full-blown shutdown of loci at the transcriptional level (piRNAs). We mainly focus on mechanisms related to piRNA and siRNA biology, two species of small RNAs that are particularly abundant in, and important for the germline. We do so mostly within the setting of (embryonic) development and are using both zebrafish and C. elegans as model systems for these studies. In addition, we aim to understand the conserved features of the mechanisms we are discovering and describing in these two model systems, by analysing small RNA pathways also in non-model animals. Finally, we have a strong interest in general germ cell development and early embryogenesis.

Maternally transmitted piRNAs are essential in zebrafish and C. elegans

Over the past few years, we have developed a transgenic system in zebrafish that allows us to study piRNA biology in healthy germ cells. Since piRNAs are essential for the proper development of germ cells, tampering with the proteins that interact with them leads to strong developmental defects that prohibit meaningful analyses at a mechanistic level. We have developed a system in which we can add or remove GFP-targeting piRNAs, while leaving the rest of the endogenous piRNAs untouched, permitting normal development of germ cells. We created this by randomly inserting transposons containing GFP sequences into a zebrafish strain that expresses GFP from another locus, selecting the strains in which GFP was silenced, and identifying the insertions that triggered silencing in those strains. Indeed, piRNAs targeting GFP are produced in these strains and they behave exactly as endogenous piRNAs. We were able to show that the system responds to the amount of target RNA: the more that is present, the stronger the silencing becomes. Furthermore, this silencing appears to be largely at the post-transcriptional level. In addition, this system has made it possible to demonstrate that the maternally provided piRNAs are required to maintain silencing. This implies that during the (early) developmental phase when these maternal products are available, key steps are made to decide which sequences will be targeted for silencing and which not. Interestingly, very similar observations we made in C. elegans, where we could even show that maternally provided piRNAs are sufficient to silence a target in the germline for the rest of the life of the worm, again strengthening the idea that during relatively early germ cell development piRNA mediated silencing is implemented, and when it is not, it will also not establish during later development.

Coordinated deposition of mRNAs into the germ cells of zebrafish

We have been studying the effect of a protein named Tdrd6 on germ cell formation in zebrafish. Maternal loss of this protein leads to less efficient germ cell specification, but the molecular reasons behind this phenotype have remained unclear. We have now been able to show, using single-cell RNA sequencing, that Tdrd6 plays a role in establishing that every germ cell that is specified receives a certain ratio of mRNA species from different germ cell-specifying genes. The way Tdrd6 seems to do this is by helping the fusion of small, RNA-protein aggregates into bigger units, such that larger aggregates of these germ cell specifying mRNAs can be loaded into the future germ cells. Interestingly, these aggregates seem to be made out of a collection of sub-granules that carries just one, or only a limited set of, mRNA species (see Figure 1). Therefore, if future germ cells inherit fragments of such aggregates that are too small, some mRNA species may be heavily under- or over-represented, possibly leading to the germ cell phenotype observed.

Future Directions

Our future work will continue to mechanistically unravel the molecular pathways that are steered by small RNA guides. We are performing a genetic screen in order to identify novel factors, and are increasingly using biochemical approaches to start to describe the mechanisms on a more molecular level. We have started to extend our studies to include structural biology, in order to be able to design specific point-mutations that disrupt specific aspects of the identified mechanisms. C. elegans and zebrafish will continue to play important roles in these studies. We will focus on how a piRNA response can be initiated de novo and want to understand how and when a transient small RNA-mediated response can be transformed into a stably inherited response. In addition, we will start to work on how small RNA pathways are connecting to other aspects of the cell's gene-regulatory programmes, including those active during germ cell specification and differentiation.