Small RNA mediated gene regulation meets phase separation

2 PhD projects offered in the IPP summer call 2021

Scientific background

Small RNA molecules are extremely potent regulators of gene expression. Research by many teams, including ours, has demonstrated that small RNAs can act in many mechanistically diverse mechanisms. However, a common theme is that many of these pathways act to silence molecular parasites, such as transposable elements, in germ cells. Strikingly, proteins acting in small RNA pathways in germ cells are nearly always found in so-called phase-separated structures. Phase separation is a phenomenon in which polymers, such as proteins and RNA, can assemble into large, often liquid-like condensates. Over the past years it has become clear that phase separation is widely spread in biological systems, but also that our understanding of regulation and function of such assemblies is very limited. We use C. elegans and zebrafish as model systems to study small RNAs and phase separation.

PhD project 1: “RNA modification and small RNA biogenesis”

The identity of the small RNAs in small RNA pathways is crucial for their target selectivity. When wrong sequences are represented, the wrong genes get silenced, with adverse effects for the organism (de Albuquerque et al). Recently, we identified a novel protein complex (Rodrigues et al.) that is required for small RNA processing in C. elegans. This complex, named PETISCO has proteins that suggest that specific modification of the 5’ Cap structure plays an important role in substrate selection. We will follow up on this work to study if indeed Cap modifications are important in the selection of small RNA precursor molecules, and how the PETISCO complex acts in the process of small RNA biogenesis. The student will use a wide variety of techniques, ranging from CRISPR mediated genome editing to high resolution microscopy and protein purification. The project will be embedded in running collaborations both within and outside Mainz.

PhD project 2: “Studying phase separation using small RNAs”

In the offered PhD project we will study how and why small RNA pathways are organized in such phase separated structures. We know of different types of condensates in both the C. elegans and zebrafish germline, and that specific small RNA-related proteins have differential affinities for different types of condensates. For instance, we recently identified a novel condensate that specifically acts during spermatogenesis to load one specific small RNA protein into mature sperm (Schreier et al.), we identified new components of known condensates that regulate their formation and activity (Placentino et al), and we identified a regulatory protein for zebrafish condensates (Roovers et al.). We will aim to understand how certain proteins can find their way in a landscape of different liquid-like condensates, how affinities of proteins for specific structures can be changed, and how this affects functionality of small RNA pathways. The project will generate new insights into small RNA-driven gene regulation, as well as into regulatory aspects of phase separation in general. Techniques that will be used are recombinant protein work, microscopy and genetics. In a collaborative setting, we will also make use of computer simulations to predict protein behavior. We are looking for a motivated student with a strong intrinsic drive to make exciting discoveries. Experience with any of the mentioned techniques is not a pre-requisite.

Publications relevant to this project

Placentino M, de Jesus Domingues A, Schreier J, Dietz S, Hellmann S, de Albuquerque B, Butter F, Ketting R (2021) Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the Caenorhabditis elegans embryo.EMBO J, 40(3):e105280

Schreier J, Dietz S, de Jesus Domingues A, Seistrup A, Nguyen D, Gleason E, Huiping L, L’Hernault S, Philips C, Butter F, Ketting R (2020) A membrane-associated condensate drives paternal epigenetic inheritance in C. elegans.BioRxiv, doi.org/10.1101/2020.12.10.417311

Rodrigues RJC, de Jesus Domingues AM, Hellmann S, Dietz S, de Albuquerque,BFM, Renz C, Ulrich HD, Sarkies P, Butter F, Ketting RF (2019) PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability.Genes Dev, doi: 10.1101/gad.322446.118

Roovers EF, Kaaij LJT, Redl S, Bronkhorst AW, Wiebrands K, de Jesus Domingues AM, Huang HY, Han CT, Riemer S, Dosch R, Salvenmoser W, Grün D, Butter F, van Oudenaarden A, Ketting RF (2018) Tdrd6a Regulates the Aggregation of Buc into Functional Subcellular Compartments that Drive Germ Cell Specification.Dev Cell, 46(3):285-301

de Albuquerque BF, Placentino M, Ketting RF (2015) Maternal piRNAs Are Essential for Germline Development following De Novo Establishment of Endo-siRNAs in Caenorhabditiselegans.Dev Cell, 34(4):448-56

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Prof. René Ketting

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