Biology of Non-coding RNA

In 1998 remarkable experiments were published describing extremely strong silencing activities of double stranded RNA. This phenomenon was named RNA interference, or RNAi, and its discovery has dramatically changed biomedical research. The discoverers, Craig Mello and Andrew Fire, were awarded the Nobel Prize in 2006. Since 1998 research by many teams, including ours, has made it absolutely clear that RNAi comes in many flavors and many different RNAi-related pathways have been identified (Figure 1). The one thing all these pathways have in common is the fact that a small, non-protein-coding RNA molecule, between 21-30 nucleotides in length, guides a protein of the so-called Argonaute family to specific RNA target molecules. Upon base-pairing between the small guide RNA and the target RNA diverse responses can be triggered, the exact outcome depending on the identity of the Argonaute (Ago) protein and most likely also on additional, largely unknown factors. As these pathways have all been recently discovered, most facets of the mechanisms involved are largely unknown. However, it is very clear that small RNAs acting in RNAi-related pathways have an immense impact on cellular function and if we want to fully understand how cells decide what to do and when to do it, it will be essential that we get a deep understanding of how these small non-coding RNA molecules have an impact on gene expression and genome organization. Below I will briefly describe the RNAi-related pathways that we are most actively studying in our team. Next to these core research lines our research interests also include other types of RNA based gene regulation and chromatin dynamics during development.

Figure 1. RNAi Pathways. For the full legend to this figure, please click here. From: Ketting, Developmental Cell, 2011

miRNAs

MicroRNAs are expressed like protein-coding genes and can display strong cell-type specificity (Figure 2). Most microRNAs are derived from relatively short RNA hairpin structures that are released from longer transcripts by the nuclear RNaseIII-type enzyme Drosha. The resulting hairpin molecule is then processed by another RNaseIII type enzyme in the cytoplasm, named Dicer. After these two cleavage steps the resulting short duplex RNA is loaded into an Ago protein, after which one of the two strands of the duplex is discarded. The resulting Ago-miRNA complex is now free to bind to mRNA targets. In animals this often involves miRNA-complementary regions in the 3’ untranslated region (UTR) of the mRNA. Recognition of an mRNA by a miRNA complex results in mRNA destabilization and translational inhibition. However, due to the relatively short regions of complementarity between miRNA and target RNA (can be as short as 7 basepairs) it is difficult to bioinformatically predict biologically relevant miRNA-mRNA interactions. We use zebrafish to experimentally define miRNA-mRNA interactions and to then better understand the biological impact of these interactions on embryonic development.

Figure 2. miRNA expression determined by in situ hybridization. The figure displays an image of a zebrafish embryo in which the expression of the miRNA miR-455b is visualized through in situ hybridization

piRNAs

The piRNA pathway is mostly germ line specific, and is essential for proper germ cell development. This small RNA driven silencing pathway mainly serves to recognize and to silence molecular parasites. It basically serves as a cell’s molecular immune system. Many of these parasites, such as transposons and retro-viruses, can integrate into the genome of its host. As such, these elements pose a threat to genome stability and thus have to be tightly controlled. Silencing of these invaders through RNAi can occur through destruction of RNA transcripts originating from them or through the packaging of their genomic copies into a transcriptionally non-productive form of chromatin: heterochromatin. Interestingly, piRNA silencing pathways are organized in discrete subcellular domains (also named nuage), often flanking the nuclear membrane (Figures 3 and 4).

Figure 3. Subcellular localization of the piRNA pathway. For the full legend to this figure, please click here. From: Ketting, Developmental Cell, 2011

It is thought that this type of organization serves to make the process more efficient and more specific. Although much progress has been made, we still understand very little about these processes. How are piRNAs exactly made? What is exactly the role of compartmentalization? How does a cell recognize self from non-self? How does the piRNA pathway trigger or maintain the formation of heterochromatin? And how are these processes integrated with the rest of the cell’s programs? We aim to answer these questions by studying the piRNA pathways in model systems like Caenorhabditis elegans (a small nematode) and the zebrafish.

Figure 4. Immune-fluorescence image showing nuage in a zebrafish germ cell. This image shows an immune-fluorescence image of a zebrafish germ cell. The various colors represent different cellular components. Green: GFP, a maker for germ cell identity. Blue: DNA. Red: Tdrd6, a component of the piRNA pathway. The aggregates of Tdrd6 protein represent nuage.