LOTUS

Figure 1: Molecular functioin of germline proteins A) Drosophila egg chamber with maturing oocyte and nurse cells (NC). Nuage surrounds the NC nuclei. Germ plasm assembles at the posterior pole of the oocyte. B) Crystal structure of the Vasa-LOTUS protein complex.

Molecular functions of germline proteins

A protein called Oskar is essential to the assembly of the germ plasm, the material that induces the formation of germ cells in some animals, and its absence has a major impact on development. We spoke to Dr Mandy Jeske about how she is combining a range of different techniques to investigate this area, alongside research into other fundamental questions around germ cell development.

LOTUS domains are found in a group of proteins known to play important roles in animal development. Based at the Biochemistry Centre at Heidelberg University, Dr Mandy Jeske leads a research group which is investigating the molecular mechanisms behind the function of these proteins. “In my group we focus on questions related to germ cell development,” she explains. One part of this research is centred on the function of these LOTUS domain proteins in animals. “In animals there are only four proteins that carry LOTUS domains. I previously studied a germline protein called Oskar that is known to carry a LOTUS domain,” says Dr Jeske. “It is wellknown that the absence of this protein leads to marked developmental defects in flies.”

Development in Drosophila

There are essentially two phenotypes to consider here, with researchers looking at the early stages of development in flies lacking Oskar. Researchers have found that eggs laid by these flies develop into embryos showing severe developmental issues. “The germ cell precursors are not produced, they are missing. Without germ cell precursors there are no germ cells, and if a larva would then develop into an adult it would be infertile,” outlines Dr Jeske. The second phenotype is that the abdominal segments usually seen on larvae are missing, resulting in developmental arrest and ultimately death. “Normally there are eight segments, and in this phenotype some or even all of them are missing,” continues Dr Jeske. “So, the function of this protein is associated with two developmental aspects, which in flies are genetically coupled – fertility and embryonic patterning.”

A lot of attention in Dr Jeske’s group is devoted to investigating how LOTUS domain proteins function during the early stages of development. It is known that Oskar is very important in assembling the germ plasm, but its molecular role is rather unclear, a topic of great interest to Dr Jeske. “We are still trying to understand the function of Oskar. We know that at the pole of the developing egg it’s required to assemble the germ plasm, and that if Oskar is missing, we see infertility and patterning defects. But beyond that, its function is not really clear,” she says. Researchers are using biochemistry techniques and X-ray crystallography to probe deeper in this area. “We need to do more biochemical studies and structural studies, using crystallography for example, to get a deeper

molecular insight into this protein in complex with interaction partners,” explains Dr Jeske.

During her post-doctoral work with Anne Ephrussi, Dr Jeske found that Oskar directly associates with a second protein. This interaction partner is an RNA helicase called Vasa, which forms a complex with Oskar, now Dr Jeske is looking to gain deeper insight. “We previously tested which part of Oskar binds to this helicase, and we found it is the LOTUS domain,” she says. Vasa itself has an unwinding activity, so it can unwind an RNA double strand to form single stranded RNA. “This process is ATP dependent – Vasa is an ATP-dependent RNA helicase,” continues Dr Jeske. “We can measure the activity of the helicase in biochemical assays – we can look at how much unwinding it does, or how much ATP it hydrolyses. When we added the LOTUS domain to the helicase, we saw a marked stimulation of this activity.”

Unfortunately, the meaning of Vasa’s unwinding activity within germ cell processes is not very well understood and neither

Transposons are like parasitic DNA sequences. Animals, plants and other organisms carry them in their genome. When transposons become active, they can effectively jump from one to another site within the genome eventually causing severe damage.

is the function of stimulation, so Dr Jeske plans further studies in future to unravel the function of Vasa’s activity. “Many steps in development are not understood on the mechanistic or molecular level,” she stresses. A second major area of research in Dr Jeske’s group centres around a regulatory mechanism called the piRNA pathway. “piRNAs are short, non-coding RNAs. They recognise RNAs which code for transposons and mediate silencing of transposon gene expression,” explains Dr Jeske. “These transposons are like parasitic DNA sequences. Animals, plants and other organisms carry them in their genome. When transposons become active, they can effectively jump from one site to another within the genome.”

Transposons

In the long term, this jumping can either serve to drive evolution. In the short term, it can cause damage to genes, which then needs to be prevented, and this piRNA-mediated defense process works slightly differently from organism to organism. Dr Jeske is again using flies to analyse the underlying mechanisms involved in the piRNA pathway. “Flies also have this piRNA pathway, and they mainly use this to repress transposons,” she says. If the piRNA pathway is not functional, then this affects the fertility of an organism, a topic that Dr Jeske is investigating in the lab. “The piRNA pathway has been mainly explored using genetic tools,” she continues. “Many proteins are involved in this piRNA pathway, and very often a defect in just a single one results in infertility.”

A number of the proteins involved here are tudor-domain containing proteins (TDRD), and in addition some have these LOTUS domains. However, it’s again not clear what their precise molecular function is. “We know that these LOTUS domains, from TDRD5 and TDRD7, similar to Oskar, bind to the Vasa helicase, and they stimulate its activity,” says Dr Jeske. A lot of attention in the wider research community is centred on the role of TDRDs and helicases in the piRNA pathway. “Most factors participating in the piRNA pathway are large, multi-domain proteins. We have developed a technique, which we are currently using to describe the physical protein-protein interaction network of the piRNA pathway,” continues Dr Jeske.

This research has already yielded some interesting insights, and has helped Dr Jeske’s team to define specific protein complexes which might be particularly important and require further analysis. The focus here is on helicase interaction partners, which can lead on to a deeper understanding of the function of these enzymes. “When we identify specific interaction partners of the helicases, we can then start to investigate the helicase activity in context of their partners,” continues Dr Jeske. The wider aim in this research is to build a deeper understanding of developmental questions on the molecular and mechanistic level, which Dr Jeske says requires a combination of biochemical research, structural studies and in vivo analyses. “We are able to combine these methods in the lab,” she says.

This means that Dr Jeske and her colleagues can explore developmental questions efficiently on the molecular level. For example, if biochemical or structural studies reveal any new insights, then Dr Jeske’s team can start to generate transgenic flies and pursue further investigation. “As soon as we observe an interesting fly phenotype, we can quickly go back to the biochemical analyses, and try to figure out a new aspect on the molecular level,” she explains.

LOTUS domain proteins in transposon silencing and translation control Project Objectives

During their life cycle, sexually reproducing organisms generate germ cells, which are key for transmission of the genome to future generations and survival of the species. In some organisms, the formation of germ cells is genetically coupled to early embryonic patterning. Recently, a novel protein domain called LOTUS was identified by bioinformatics, which is highly conserved. In animals, LOTUS domain proteins are essential germline factors. It has also been established that the piRNA pathway contributes to genome stability of the species. However, the molecular properties of LOTUS domain proteins and their specific roles within the control of translation and the piRNA synthesis pathway remains poorly understood. The aim of Dr Jeske’s research is to structurally and functionally characterize this novel and essential protein family to provide mechanistic insight into important but poorly understood aspects of post-transcriptional control of gene expression in the germline.

Project Funding

Deutsche Forschungsgemeinschaft (DFG) - Project number 377227242

Contact Details

Project Coordinator, Dr. Mandy Jeske Heidelberg University Biochemistry Center (BZH) Im Neuenheimer Feld 328 D-69120 Heidelberg T: +49-6221-544728 E: jeske@bzh.uni-heidelberg.de W: https://www.bzh.uni-heidelberg.de/jeske

Kubíková, J., Reinig, R., Salgania, H.K., and Jeske, M. (2020). LOTUS-domain proteins - developmental effectors from a molecular perspective. Biol. Chem. 402: 7 - 23. Jeske M., Müller C.W., Ephrussi A. (2017). The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage. Genes. Dev. 31(9): 939-952.

Dr Mandy Jeske

Dr Mandy Jeske is a Junior Research Group Leader at Heidelberg University Biochemistry Center in Germany. She gained her degree at Martin-Luther-University Halle-Wittenberg, then held a postdoc position at the EMBL Heidelberg before taking on her current role. She holds an Emmy Noether Fellowship funded by the DFG.