AP1: Ribosome biogenesis in Archaea | S. Ferreira-Cerca
Ribosomes are evolutionary conserved macromolecular machines involved in protein synthesis. Given their pivotal role in gene expression, cellular growth and proliferation, the detailed molecular understanding of ribosomes function and how they are biosynthesized are fundamental questions in biology.
Eubacterial and eukaryotic ribosome biogenesis have been extensively analyzed during the last decades. However our understanding of the archaeal ribosome biogenesis pathway remains relatively scarce. In order to fill this gap and shed light on this fundamental process, we aim to systematically analyze the archaeal in vivo ribosome biogenesis pathway.
To this end we are using a combination of functional genomic and proteomic approaches in order to discover the archaeal ribosome biogenesis pathway using the advantage of two genetically tractable organisms: the halophile euryarchaeon Haloferax volcannii and the thermo-acidophile crenoarchaeon Sulfolobus acidocaldarius as model organisms.
As a proof of principle we have already generated mutants of putative archaeal ribosome biogenesis factors conserved either in bacterial and/ or in eukaryotic cells and are currently analyzing their putative role(s) in the archaeal ribosome biogenesis pathway in vivo. Our preliminary work already suggest that the archaeal ribosome biogenesis assembly pathway is characterized by the usage of a mixture of bacterial- and eukaryotic-like features that are complemented by additional Archaea specific features.
Overall our work will help to better characterize the in vivo principles of RNP assembly in Archaea. Thereby providing among others; a conceptual framework for the understanding of key evolutionary conserved principles of ribosome assembly, and a molecular understanding of the gain of functions and/ specialization of the archaeal ribosome biogenesis pathway.

AP2: Structural and functional analysis of early acting ribosome biogenesis factors | J. Perez-Fernandez
Ribosome biogenesis is an energetic expensive process which requires the action of more than 150 ribosome biogenesis factors (hereafter called auxiliary factors) conserved between yeast and humans. Some auxiliary factors form protein subcomplexes (called UTP) which are also conserved through evolution. The inefficient production of fully active ribosomes seems to be the specific cause of a new set of diseases grouped as ribosomopathies. Among the mutations found in those diseases, some are located in genes coding for auxiliary factors but the molecular explanation of their effects remains elusive. In this regard, the advanced knowledge of the yeast ribosome biogenesis process is a clear advantage to understand this process in higher eukaryotes. Recently, we have described the in vitro reconstitution of yeast UTPs which is a valuable tool for functional studies and high resolution structural analysis. Moreover, the results suggest that assembly of UTPs occurs independently of ongoing ribosomal production. The aim of this project is to characterize: i) the in vivo formation of UTPs, ii) The structure and function of the different UTP components and iii) The molecular effects of mutations found on genes coding for UTP components.

AP3: Identification and characterisation of components associated with the archaeal transcription machinery | W. Hausner
The archaeal transcription system is a more simplified version of the eukaryotic one and therefore provides a kind of facilitated access for studying some details of the transcription process. Our model organism is Pyrococcus furiosus, which belongs to the hyperthermophilic euryarchaeota. This organism is genetically tractable and in addition the archaeal RNA polymerase can be completely reconstituted from recombinant components. We have also established the ChIP-Seq technique for this organism and a genome size below 2 Mbp provides excellent conditions for global approaches.

The main focus of the prosposal will be on the identification of additional components which interact with the basic transcription machinery during initiation or elongation. One of these components is the replication protein A, a single stranded DNA binding protein. In cooperation with the group of Didier Flament (University of Brest, France) we could demonstrate that this protein stimulates archaeal transcription in vitro. Recent in vivo data also indicate a role of this protein in eukaryotic transcription. As the functional details of this protein are not known, we would like to analyse the role of RPA during transcription. We have also evidence that Rad25, a helicase which is in Eukarya a part of TFIIH, is in contact with the archaeal RNA polymerase. The function in archaeal transcription is not known. Furthermore, we are interested to investigate the composition of transcription complexes transcribing protein or ribosomal genes. Due to the prokaryotic nature it is most likely that the archaeal Spt4/Spt5 complex combines transcription with translation, but if Spt4/5 is also involved in rRNA transcription is still an open question. To answer this question we would like to establish a procedure which allows the isolation of gene-specific complexes from formaldehyde fixed cells.

AP4: Nucleolar control of genome organization and function | A. Németh
The nucleolus is the site of ribosomal RNA transcription and ribosome biogenesis, and facilitates a number of other cellular processes like cell cycle progression, stress sensing and RNA modification. Although it is the largest compartment of the cell’s nucleus, its role in genome organization and function is poorly understood.
Nucleolus-associated chromosome domains were identified genome-wide in human cells (Németh et al., 2010, van Koningsbruggen et al., 2010) providing a snapshot of genome organization around the nucleolus. Yet, questions about the dynamics of the spatial arrangement of this part of the genome remained to be answered. Since the nuclear and nucleolar architecture get largely remodeled during cellular aging, we aim to systematically analyze nucleolus-associated chromatin reorganization in this fundamental biological process.
To this end, we will make use a combination of functional genomics and DNA-combing-based single-molecule analyses approaches, which were recently developed in our laboratory. These investigations will be complemented with conventional immunofluorescence analyses, 3D-immuno-FISH experiments, quantitative RT-qPCR and northern blot analyses to reveal the exact sequence of alterations in chromatin structure, DNA modification patterns, transcriptional activity and nuclear organization of specific nucleolus-associated chromosome domains during cellular senescence.In the initial phase of the project nucleoli of IMR90 human lung embryonic fibroblasts were isolated from a young, proliferating cell population, as well as from senescent cells. Nucleolus-associated DNA was analyzed on high-resolution microarrays by comparative genomic hybridization. Additionally, RNA was extracted from the same cell populations and subjected to global gene expression analysis. The results indicate the role of the nucleolus-associated chromatin in genome reorganization and gene regulation during senescence. Furthermore, based on the results of the high-throughput analyses we could select specific chromosomal domains and chromatin regulator proteins, which we aim to analyze in detail in subsequent experiments. In summary, our investigations will provide novel insights into the spatial and transcriptional dynamics of the genome during cellular aging and shed light on the role of the nucleolus, the largest RNP-producing compartment of the nucleus, in nuclear organization and function.

AP5: Structure determination of the dsRNA-transporting channel SID-1 involved in RNAi in C. elegans by single particle cryo-EM | C. Ziegler
Single particle (SP) cryo-EM has achieved lately near-atomic resolution for asymmetric and dynamic protein assemblies. This breakthrough towards atomic resolution was accomplished by improvements in microscope design and fast digital cameras detecting electrons directly. In addition new image processing algorithm compensate for resolution-limiting movements of proteins caused by the electron beam. Recently also atomic structures of membrane proteins could be solved by SP cryo-EM, e.g., the TRPV1 ion channel in distinct conformations. We will use state-of-the-art SP cryo-EM to determine the structure of the highly conserved dsRNA-gated and dsRNA-transporting channel SID-1 from C. elegans. This channel is required for the import of RNAi triggers in non-neuronal tissues in C. elegans where RNAi is remarkably potent. Recent SAXS studies on the extracellular domain of SID-1 have revealed a tetrameric assembly with a central pore in the dimensions of dsRNA. We will express SID-1 as well as structural and functional mutants in the baculovirus system and reconstitute the channel in amphiphilic detergents or nanodiscs. Moreover, we will study its regulatory interaction with SID-2, a small single-pass membrane protein that is expressed in gut cells where it interacts most likely with ingested dsRNA. The mechanisms and regulation of dsRNA import mediated by SID-1/SID-2 and its role in RNAi in C. elegans will provide new perspectives for optimizing RNAi in other species. Furthermore, we will exploit x-ray crystallography and NMR to investigate structure and dynamics of the extracellular domains that are suggested to control the access of dsRNA. In addition, we will provide our knowledge on state-of-the-art cryo-EM also to other groups, e.g., we will in collaboration with Dr. Philip Milkereit (University Regensburg, Biochemistry III) apply SP cryo-EM to investigate the structure of different ribosome assembly intermediates from Saccharomyces cerevisiae in complex with their respective assembly factors.